Brahl Research, 140 (1978) 15-32 ,~©Elsevier/North-Holland Biomedical Press

15

HORSERADISH PEROXIDASE AS A R E T R O G R A D E L Y - T R A N S P O R T E D , DETAILED DENDRITIC MARKER

DONALD A. KEEFER* Max-P#mck-hlstitute J~ir Hirnforschung, Neurobiology Department, 6 Frankfurt/M.-Niederrad (G.F.R.)

(Accepted April 27th, 1977)

SUMMARY A method is described that produces detailed 'Golgi-likc' staining in neurons following retrograde transport of horseradish peroxidase (HRP). Of 4 commercially available HRP preparations, which were compared for their ability to produce solid neuronal staining in certain regions of the rabbit thalamus 48 h after striatal injection, two preparations (Miles Laboratories and Serva Biochemical) were found to be most efficient. When dimethylsulfoxide (DMSO) was used as the solvent for the injected HRP, a significant increase in the number of solidly labeled neurons in the thalamus was realized. Approximately 2 % DMSO produced the greatest increase when compared with water as the solvent. Dendritic detail visualized by this method is shown to compare favorably with that produced by Golgi staining.

INTRODUCTION When detailed dendritic pictures of neurons are desired at the light microscopic level, the technical arsenal available to the neuroanatomist has, until recently, been limited to the various modifications of the century-old Golgi reduced silver methods v, 11,12,16,19,35,51. However, its capriciousness and lack of selectivity have somewhat limited its application. Several variations of the extracellular iontophoretic and intracellular injection techniques have been employed recently to study axonal and dendritic patterns of single neurons17,28,31,41,4~,5°,53 5a,ss. These latter methods, by virtue of their ultimate selectivity, are vital to studies bridging the interface between the electrophysiological, histological and hodological properties of neurons, but are of limited value when larger populations of neurons need to be studied. * Present address: Department of Anatomy, University of Virginia School of Medicine, Jordan Building, Charlottesville, Va. 22901, U.S.A.

16 The method which employs horseradish peroxidase (HRP) as a retrogradelytransported marker substance has allowed neuroanatomists to trace afferent connections to specific neural regions, but, unfortunately, has not concomitantly provided the degree of dendritic resolution available with the Golgi or intracellular injection methods. Two general types of intracellular HRP localization have been noted with the existing HRP method 26,27,4a,4v. The granular type (gHRP) is most characteristic of the retrogradely-transported H RP where lysosome-sized granules fill the cytoplasm of the cell body to varying degrees and often extend for some distance into the proximal dendrites. A second type of localization, usually referred to as 'agranular' or diffuse localization has been described in the periphery of the injection site or occasionally more distantly and results in a neuron containing homogeneously stained cytoplasm extending into secondary and even tertiary branches of larger dendrites. However, as shown by others 13,~ and detailed in this paper, the diffuse localization is often accompanied by granular HRP. Therefore, these two types of localization shall be distinguished here by using the term ~diffuse' HRP neuron (dHRP) to indicate those neurons which contain only homogeneously stained cytoplasm5 The term 'diffuse-granular' HRP neuron (dgHRP) will designate the neurons which contain both diffusely stained cytoplasm and darkly stained granules extending only into proximal dendrites. In this paper, yet another type of HRP-marked neuron, called "solid" HRP neuron (sHRP), will be described. This neuron is seen both near the injection site and interspersed among other retrogradely-labeled gHRP and dgHRP neurons afferent to and distant from the injection site (Fig. 7). These sHRP neurons send their axons to or through the site of injection, and contain varying numbers of large stained granules distributed within an opaque or darkly translucent cytoplasm. The sHRP neurons diffel from the previously described neurons in that tile HRP homogeneously fills the entire cell, extending even into the distal-most portions of its dendritic processes and their spines. This results in a 'Golgi-like" appearance of the sHRP neurons ~. In this paper the experiments leading up to this modified HRP technique are described and a preliminary assessment of its usefulness as a detailed dendritic marker at the light microscopic level is offered. While describing the procedure used i~l obtaining sHRP-labeled neurons, this report is not intended to specify a final recipe, but rather to present certain preliminao' observations which are worth further exploration and which may become the basis of a valuable adjunct to the Golgi methods. MATERIAL AND METHODS Experiment I Four commercially available horseradish peroxidase preparations were examined: Sigma (Sg, Germany) H R P type II, Sigma HRP type VI, Serva (St, Germany) HRP, and Miles (MI, England) HRP. The enzymes were purchased between June and November 1975 and stored for less than two weeks according to instructions accompanying the material. Bilateral striatal injections of 30 ~,/, H R P in 0.4 #1 distilled water were made stereotaxically in 7 female rabbits weighing 1.0-1.4 kg. For direct compa-

17 TABLE 1 Mean number o f sHRP neurons (first value) and g H R P neurons (in parentheses) in 32-:m7 thick sections Of the thMamus o f rabbit 48 h after an ipsilateral striatal injection o f 0.4 1d 30 % H R P /?ore Sigma Chemical Co. (Sgll, Sg VI), Serva Biochemical Co. (Sr) and Miles Laboratories Ltd. (MI)

Values represent cell counts made from 10 to 20 coronal sections throughout the thalamus. Fixative

Animal no.

Paraformaldehydeglutaraldehyde

K084 K086 K087 K093

Formaldehyde

K085 K089 K091

Sgll

Sg VI

0.0 (0.9)

Sr

2.8 (33.0)

0.0 (34.3) 29.2 ( : 109)* 0.0 (14.3) 0.0 (0.8) 0.0 (0.0) 0.0 (0.2)

Ml

18.6 (> 200)* 30.2( , 127)* 12.7 ( 65)*

0.0 (28.4) 0.0 (19.6) 9.3 (23.0)

* When large numbers of gHRP neurons appeared in a single section their number was estimated as '100, 150, 200 etc.' rison, a different H R P was injected into each side of the brain (Table I). The injection was administered over 10 m;n and the cannula was left in place for an additional 10 min. The cannula consisted of a drawn glass capillary tube with an outside diameter at the tip o f approximately 100/~m, attached to a 1.0-/d Hamilton syringe. After 48 h, each rabbit was perfused transcardially with either 1 0 ~ unbuffered formaldehyde or 2'!1~ paraformaldehyde-l.5~o glutaraldehyde in 0.01 M phosphate buffered saline (PBS) ae. The brain was removed quickly and soaked in its respective fixative overnight and then transferred to PBS with 3 0 ~ glucose for 2-4 days. The brains were fi'ozen in COe and a I in 3 series of 32-#m thick sections were cut. The sections were placed into 0.075'~o 3,Y-diaminobenzidine tetrahydrochloride (DAB) in Tris buffer (0.1 M , pH 7.4) at room temperature20, 47. After 10-20 rain, 0.006 °£, hydrogen peroxide was added and the staining reaction was continued, with occasional stirring, for 40 rain. The sections were then washed in Tris buffer, stained in cresyl violet and mounted. Solid H R P and g H R P neurons in each side of the thalamus were counted. Experiment II

Adult female rabbits (1.0-1.6 kg body weight) received bilateral striatal injections o f 0.2-/d 300/00 MI H R P unless otherwise indicated. (A) In 4 rabbits, each side was injected with H R P dissolved in either 0.2, 2.0, 20.0 or 1 0 0 ~ D M S O in water. (B) In 5 rabbits, each side was injected with H R P dissolved either in distilled water or in 2.0 o/ DMSO. Serial coronal sections alternating in thickness of 32, 64 or 96 # m were taken through the thalamus, stained with DAB and counterstained with cresyl violet, either with or without osmium intensification. The numbers of s H R P and g H R P neurons were counted in representative sections on each side of the thalamus. Finally, neurons from rabbit thalamus were stained by a modification of the Golgi method o f Colonnier 9 and Valverde 60. Neurons stained by the Golgi method were then compared with the s H R P neurons obtained in Experiments I and II. A

18

F

Fig. 1. The striatal injection site of H R P after 48 h survival period. A: injection of 0:2 14 30% MI H R P in head of caudate nucleus showing center of injection site with localized tissue disruption and periphery of injection with brown staining of neuropil. Some diffusely stained axons extend toward the internal capsule (IC). x 250. B: detail of edge of injection site seen in A showing portions of spiny dendrites. × 700. C: a sHRP neuron with spines in periphery of injection site in medial caudate. x 250. D : several sHRP neurons in periphery of striatal injection site in putamen. Injection center is to the left. × 150. E: a dgHRP neuron in periphery of injection site in caudate, x 400. F: camera lucida drawing of neuron with spines from periphery of H R P injection site in caudate. × 900.

19 450-nm and/or 475-nm narrow band interference filter (Schot@ 6 was used when examining HRP-labeled neurons. RESULTS Injection site After 48 h, all injection sites consist of a central region immediately surrounding the tip of the cannula tract (approx. 0.3 x 1.5 ram) which displays opaque brown staining with evidence of cellular disruption and presence of erythrocytes and a peripheral region consisting of lighter staining neuropil-containing gHRP, dgHRP and (with Sr and M1 HRP) sHRP neurons with glia and pericytes containing large HRP granules and/or diffuse brown cytoplasm (Fig. 1). Use o f20°//o or 100~ DMSO makes the injection site appear smaller. The shape of the injection site is often made irregular by passing fiber bundles. Very few, if any, 'agranular' or dHRP neurons can be conclusively identified near the injection site at this survival time. Most of the stained neurons are stained in a granular (gHRP) or diffuse-granular (dgHRP) manner regardless of the type of HRP injected (Figs I E). Diffusely stained axons are seen emanating fi'om the injection site regardless of the type of HRP used, but they can be followed for a longer distance in preparations injected with Sr or M1 HRP than in preparations with Sg HRP. The axons in the former case can often be traced en masse to the neocortex and midbrain levels and individually throughout the thalamus. Thalamus Forty-eight hours after injection of M1 or Sr HRP into the rabbit striatum, gHRP, dgHRP and sHRP neurons are distributed extensively within selective regions of the neocortex, thalamus and substantia nigra, and sparsely within the nucleus raphe and nucleus subthalamicus. The localization within the neocortex has been reported elsewhere a'~. Only the thalamus will be evaluated in this report. Although the quantity and staining quality of labeled neurons varies with the parameters of H RP administration, the overall thalamic distribution of labeled neurons is consistent (Fig. 2). The majority of the labeled neurons is situated within or adjacent to the intralaminar nuclei and the centromedian-parafascicular complex. The concentration along the internal medullary lamina is especially pronounced. Labeled neurons extend outward from the borders of these intralaminar and parafascicular accumulations and are seen in varying numbers in regions containing neurons thought to project primarily to the cortex. These regions include the three anterior nuclei, the midline nuclei, with the exception of the paramedian nucleus, the laterodorsal and lateroposterior nuclei, and the mediodorsal, ventrobasal and medial ventroposterior nuclei. When these nuclei border on the intralaminar and parafascicular concentrations, the number of labeled neurons is usually highest in that part of the nucleus immediately adjacent to the intralaminar or parafascicular regions. Fixation of the tissue with 2 °//,, paraformaldehyde-l.5°/o glutaraldehyde (PG) allows for staining of a much larger number of sHRP and gHRP neurons than does 10 ~ formalin fixation (Table I). Additionally, the resolution of the stained dendrites

20 K150

K151

V

K150

* "'*

,*

• tJ:,;*.;" am v "t':, .:

"'

v.e

0!

K151

Fig. 2. Distribution of sHRP neurons (stars) and gHRP neurons (dots) at two thalamic levels m t~xo rabbits (K150 and K151) 48 h after striatal injections of 0.2 ld 30% MI HRP. On the left side the injected HRP was dissolved in 2% DMSO, on the right, in water. HRP-labeled neurons are in similar areas on both sides of the thalamus but more sHRP neurons appear on the left. The densest accumulations of labeled neurons are generally associated with the internal medullary lamina and the centromedian-parafascicular complex. The distribution was determined from 64-/~m thick coronal sections. Schematics adapted from Gerhard TM, Abbreviations: ad, n. anterior dorsalis; am, n. anterior medialis; av, n. anterior ventralis; f, fornix; m, t. mammillothalamicus; P, centromedian-parafascicular complex; pc, crus cerebri ; r, n. rhomboideus, s, stria medullaris; st, n. subtbalamicus; th, t. habenulointereruralis; to, t. opticus; v, n. ventralis.

is finer with the P G fixation t h a n with formalin fixation. Regardless of the fixative used, Sr a n d M1 H R P , in contrast to Sg H R P , result in greater n u m b e r s of g H R P and d g H R P n e u r o n s visible in the thalamus, p r o d u c i n g a complete spectrum of labeling, r a n g i n g from g H R P and d g H R P n e u r o n s to s H R P neurons, totally filled with H R P (Figs. 3 and 4). In the present experiments, s H R P n e u r o n s are seen only when Sr or M1 H R P are used. The s H R P n e u r o n s generally comprise 10-30 ,°i; of the labeled n e u r o n s when a 0.4-,ul injection is made a n d up to 50 or 60% when a 0.2-/tl injection is made, possibly suggesting an i n v o l v e m e n t of axon damage in s H R P n e u r o n production. The difference between the n u m b e r of s H R P n e u r o n s shown for MI a n d Sr H R P in Table I and those shown in Table II u n d e r 'distilled water' p r o b a b l y reflects the different volumes of H R P injected: 0 . 4 / t l in the first case a n d 0.2 #l in the latter. W h e n D M S O is used as a solvent instead of water, significantly more s H R P n e u r o n s

21

Fig. 3. Various types of n e u r o n a l staining seen in the t h a l a m u s of the rabbit 48 h after striatal injection of 30 % Ml or Sr H R P in water or 2 ~ D MSO. A - E : examples of the gradient o f staining types seen within the g H R P (A) a n d d g H R P (B E) classifications, y, 700. F: a s H R P n e u r o n in the n. lateralis magnocel[ularis, x 400. G : a s H R P neuron at the lateral edge of the n. centralis lateralis. 175. A c a m e r a lucida drawing of this n e u r o n is seen in Fig. 5. H : two s H R P n e u r o n s in the n. r h o m b o i d e u s (a midline nucleus). × 250. Staining of especially fine caliber dendrites was slightly improved with o s m i u m intensification. F H are counterstained with cresyl violet. F was p h o t o g r a p h e d without interference filter.

Fig. 4. Examples of s H R P neurons in the thalamus of the rabbit 48 h after striatal injeciion oi" M1 or Sr H R P dissolved in water or 2 °i; DMSO. A: p h o t o m o n t a g e showing the dendritic arb~wization aild proximal axon of a neuron in the n. anterior medialis immediately adjacent to the n. centralis medians, The n. r h o m b o i d e u s (top) in this animal shows the heaviest g H R P labeling seen in these expcriments. At the lower left is a s H R P neuron near the border of the n. ventralis and n u m e r o u s diffusely stained axons can be seen entering the thalamus. 200. B: a s H R P neuron in the n. ventralis meclialis. 200. C: a s H R P n e u r o n in the n. centralis medialis. 200. D: a s H R P neuron near the dors:~,l border of, the n. ventralis medialis - 250. E: a s H R P neuron in the n. lateralis magnocellulaH~ 250, I-: ~ s H R P n e u r o n near the dorsal border of the n. anterior ventralis. 250. Sections B- I-: were counterstained with cresyl violet. B and F were p h o t o g r a p h e d without an interference filter.

23 TABLE II Mean number o f s H R P neurons in 32 I~m thick coronal sections in the thalanms o f rabbit 48 h after an ipsilateral striatal #zjection o f 0.2/~l 30 °o M I H R P dissolved ill distilled water or in various concentrations o f D M S O

Values represent cell counts made from 10 to 20 coronal sections throughout the thalamus. Counts made from 64-/tm and 96-1~mthick sections are adjusted to 32-1tin equivalency. Erp. no.

IIA

liB

AtthtTal no.

Distilled water Per cent D M S O as H R P solvent

KI27 KI28 K152 K153 KI45 KI46 K150 KI51

0.2 o;

2.0 %

3.3

6.9

11.2 3.4 4.7 4.3 5.4 2.1

15.4 5.9 8.4 8.3

Ratio

20.0 °o

/ 000;

5.1" 7.0"

0.1" 1.5" 1:3.3 1:1.4 1:1.6 1:4.0

* Quality of staining is generally poor. are seen in the thalamus (Table II). The increase in the number of labeled neurons in individual animals injected with H R P in 2~; DMSO rather than water varies from 27 of to nearly 300'~ with the mean increase being more than 2.3-fold. Of the 4 DMSO concentrations tested (Experiment llA), 2 ~o is judged to be generally the best, since it provides a substantial quantitative increase in sHRP neurons without affecting the quality of distant neuronal staining. DMSO at concentrations of 20 }o~ or 100 3o produced few sHRP neurons, Although DMSO greatly increases the number of sHRP neurons, the increase in the number of g H R P neurons is relatively small. Finally, the quantity and quality of intbrmation which may be derived from the neuromorphological picture of sHRP neurons compares favorably with that provided by the Golgi technique (Fig. 5). Both methods reveal dendritic arborizations to a similar extent and, additionally, provide sufficient resolution to visualize even spinous processes (Fig. 6). Solid HRP-stained dendrites usually appear finer than Golgi-stained dendrites and are occasionally more difficult to follow. Tissue sections 64 and 96 #m thick proved to be more useful than 32-~m thick sections since the latter generally contain less of the total dendritic arborization. Counterstaining with cresyl violet does not increase the difficulty of finding or resolving the sH RP neurons if the narrow band interference filter is used in the microscope optics, since this essentially eliminates visualization of Nissl-stained cells. Additionally, the short wavelength of the transmitted light provides increased resolution of fine processes. DISCUSSION The thalamic distribution of labeled neurons corresponds very closely with that reported by Kuypers et al. 39 and by Nauta et al. 47 in the rat following H R P injections

24

f J

)

/

(

? f

f

a

J

t

//)

Fig. 5. Camera lucida drawing of a sHRP neuron at the lateral edge ef the n. centralis lateralis of rabbit 48 h after striatal injection of MI HRP in 2% DMSO. A photograph of this neuron is seen in Fig. 3G.

in the caudato-putamen. When injections were confined to the caudato-putamen, the latter group similarly found that the majority of labeled neurons are associated with, but are not limited to, the loosely-defined intralaminar and centromedian-parafascicular areas, especially along the internal medullary lamina. The present results indicate that these neurons may be labeled both as a result of uptake by intact or damaged axon terminals in the striatum and possibly also by fibers of passage in the internal capsule. The neurons of the centromedian-parafascicular complex and intralaminar areas are thought to project, respectively, to the putamen and the caudate t°,z3,4's,47,~l .

Fig. 6. A and B. Details of s H R P n e u r o n s in the t h a l a m u s of the rabbit 48 h after striatal injection of H R P in 2 ~o D M S O . A : p h o t o m o n t a g e showing an intralaminar s H R P n e u r o n a n d a portion of its spiny dendritic processes within the lamina which separates the anterior dorsal a n d anterior ventral thalamic nuclei. Several neurons within the ventral border o f the anterior dorsal nucleus are seen along the top of the photo. A small pericyte containing large granules is seen a r o u n d capillaries in the lower right a n d a g H R P neuron overlies the descending dendritic process. × 600. B: high magnification p h o t o m o n t a g e of a portion of the dendritic tree of a s H R P neuron in the n. centralis lateralis. Spinous processes appear as knoblike- protrusions at points along the dendrite, x 900. C a n d D: details o f n e u r o n s in the rabbit t h a l a m u s as revealed by the Golgi m e t h o d of Colonnier. C: photom o n t a g e o f a Golgi-stained neuron a n d one of its dendrites located in or near the region between the a n t e r i o r d o r s a l a n d anterior ventral nuclei. C o m p a r e the staining and resolution with that in A above.

26 Consequently, the large numbers of sHRP and gHRP neurons seen in these nuclei probably result from uptake by their intact or damaged axon terminals. Since, on the other hand, some g H R P and sHRP neurons are also present in other thalamic nuclei which are known to project primarily to the cortex via the internal capsule10, 47, it is quite possible that labeling of these neurons results from axonal uptake of the HRP by some internal capsule fibers. However, since the percentage of solidly labeled neurons in these other nuclei is generally small and their atrophy could easily have escaped detection with degeneration techniques, it cannot be ruled out that these small subpopulations of labeled neurons in areas adjacent to the parafasciculm and intralaminar areas may also terminate directly, or via collaterals, in the striatum. Diffuse H R P component. The precise mechanism by which sHRP neurons are produced is unknown. However, all 4 types of HRP staining, vie gHRP, dHRP, dgHRP and sHRP, appear to consist of quantitative variations of the lwo basic staining components, diffuse and granular HRP. Thus, the distinction between e.g. dgHRP and sHRP neurons may simply be a matter of the extent of somal and dendritic filling with each H R P component. Proposed causes for this form of uptake have been attributed to poor fixation of the tissue, mechanical damage to the plasmalemma, or membrane damage resulting from the excessive extracellular concentrations of the protein 2,4,s,29,a°,4s,sT. In the present experiments poor fixation is not likely to be responsible for producing the diffuse component since all signs at the LM and EM levels 27 indicate that the tissue is well fixed. It is uncertain to what extent, if any. mechanical damage to the axon, caused by the injection, is responsible lk)r the occurrence of the diffuse staining component. However, neurons containing diffuse HRP have been described in the brain following both intravenous and topical applications of H R P which circumvent or minimize mechanical damage to the neuronsl, ;~;~.Additionally, studies of H R P accumulation following uptake in crushedZ~,a~,:~7, :~s and cut ~. ~4 axons indicate that, while axons emanating from the HRP application site may be

Fig. 7. Schematic representation of the 4 general types of HRP staining seen in neurons after HRP injection. See text for explanation.

27 diffusely stained, retrogradely-transported intrasomal H R P is exclusively granular and is indistinguishable from H R P taken up via intact axon terminals (however, see ref. 1). On the other hand, the present data are compatible with, but do not prove, the hypothesis that the diffuse staining component seen in dHRP, d g H R P and sHRP neurons results from membrane alteration or damage caused by high extracellular H R P levels. Diffuse uptake of protein by neurons, seen after administration of high protein concentrations, has been attributed to cell damage2, 4. Turner and Harris 5:~, for example, suggest that cell damage is responsible for the diffusely stained neurons that they describe in the cortex three hours after topical application of HRP. Broadwell and Brightman 4 similarly describe diffusely stained neurons in the hypothalamus 2 4 h after intravenous administration of Sg HRP, but find that this diffuse labeling disappears after 6 h. Further evidence suggests the possibility that transient membrane alterations, rather than damage per se, may be the cause of diffuse HRP uptake. After applying H R P to one or more neurons by discrete extracellular iontophoretic ejection, while recording, presumably, from one of the filled neurons, Lynch et al. 4t report that the electrophysiological activity of the neuron decreases only transiently during the ejection process and apparently resumes normal electrochemical activity at a time when large amounts of diffuse H R P have moved into distal recesses of its dendritic tree. Finally, it is apparent from the present experiment that d g H R P and sHRP neurons loaded with diffuse H R P are still capable of either packaging the diffuse intracellular H RP or taking up 'normal' amounts of granular HRP, presumably by endocytosis. Granular HRP component. Characteristics of the granular component of H R P staining, present in gHRP, dgHRP, and sHRP neurons, are more clearly established. Ultrastructural studies describe HRP-filled, membrane-bound vesicles forming at the plasmalemma by pinocytosis when the adjacent extracellular space is filled with free HRP. This endocytotic formation of HRP-filled vesicles occurs primarily at the axon terminal, but also takes place along the intact, crushed or cut axon, in the perikaryon and along the dendrites(;,14,'-)2,25,a~;-as,40,57,~9. When taken up at the axon terminal region, H R P is transported retrogradely along the axon at approximately 50 #m/min in the form of small vesicles or saccules which are taken up by lysosomes or which coalesce to form lysosome-sized bodies once in the perikaryon:31, 40. HRP differences. The reasons for the differences in staining characteristics between MI and Sr HRP, on the one hand, and Sgll and SgVI H R P on the other, is uncertain. Spatz a~ noted that Sr H RP injected into cortex resulted in a greater number of more intensely stained gHRP-labelled cortical neurons than did the use of SgVI HRP. This has been confirmed in the present sludy. This increased staining of retrograde[y-labeled neurons seen with Sr and M1 H R P applies to both the number and intensity of staining of gHRP-laheled neurons and to the greatly increased occurrence of s H R P n~urons. Although there are several plausible explanations for the staining differences seen among the 4 HRP preparations, the one currently under investigation is that the Sr and MI H R P may appear more potent because of a prolonged enzymatic integrity. That is, Sr and MI HRP, both in granular and diffuse form, ,nay be more

28 resistant to the enzymatic degradation and inactivation processes occurring within the cell so that, subsequently, sufficient concentrations of the HRP could reach the distant recesses of the cell by active transport and/or diffusion before it is enzymatically inactivated. The reason for this is still uncertain but may reside either in the fact that the Sr and MI HRPs are of higher enzymatic purity or that the HRPs are of different isoenzymic composition. In this latter regard, Bunt et al. ~, have shown that three isoenzymes of HRP greatly differ in their ability to retrogradely label neurons. The active and inactive isoenzymes differ significantly both in terms of amino acid composition (and isoelectric points) and in carbohydrate composition. Consequently, it is feasible that different isoenzymic compositions may occur within various H R P preparations and that configurational differences among isoenzymes, unrelated to their enzymatic activity, may render them differentially susceptible to inactivation by cellular mechanisms. In agreement with Bunt et al. 8 the relative enzymatic activities of the 4 HRP preparations examined here {as reported by the suppliers) do not correspond to the observed differences in staining characteristics. The specific enzyme activity, in Purpurogallin Units, for the Sgll, SgVi, M1 and Sr HRP preparations are 185, 289, 237 and 250, respectively. On the other hand, the enzymatic purits,, or RZ value, for the Sgll, SgVI, MI and Sr HRP are 1.3, 2.65.3.1 and 3.0, respectively, and thus correspond generally with observed staining differences. The higher purity may be associated with a higher percentage of a particular isoenzyme, which in turn possesses a configurational arrangement rendering it a longer intracellular half-life. Alternatively, the manufacturer's purification process may introduce some contaminant which may depolarize or otherwise alter the neuronal membrane so as to facilitate HRP uptake. Dimethyls'ulfoxide. The role of the DMSO in producing the larger number of sHRP neurons in the thalamus probably results from its well established function in increasing the permeability of cell membranes a (see also ref. 46). If the DMSO causes the axonal membrane to become more permeable to the extracellular H RP, this may increase the number of axons and terminals near the injection site which become filled with diffuse HRP and also increase the total amount of diffuse HRP which enters the axon prior to retrograde transport. By increasing the axonal membrane permeability during the early moments following injection, while the extracellular concentration of free HRP is highest, more HRP molecules would have the potential to enter the axon, presumably by diffusion. However, since DMSO does not alter the thalamic distribution of labeled neurons, it would not appear to increase HtRP entry into myelinated axons of passage in the internal capsule. By an unknown mechanism, the DMSO also appears to exert a smaller, but still significant, increase in gHRP uptake, thereby increasing the number of retrogradely-labeled gHRP neurons. Finally, Donoso et al. 14 have recently reported that, while 2% DMSO has no discernible effect on fast axoplasmic transport in vitro, higher concentrations exert a stabilizing action on microtubules, thereby blocking fast axoplasmic transport, Although not directly comparable to the present study, this data may explain the decrease in retro~ gradely-labeled neurons seen with the 20 and 100 ~0/oDMSO preparations. H R P vs. Golgi. The granular label of neurons with HRP provides the investi-

29 gator with topographical data concerning the neurons afferent to the injection site. Diffuse granular labeling may additionally allow general identification of the class of cell labeled (e.g., stellate, pyramidal, fusiform etc.) especially at particular developmental stages 27. When a neuron is solidly labeled with HRP one knows not only where the neuron projects (within limits) but also has a relatively complete picture of its dendritic configuration. This solid HRP method compares favorably with the Golgi-reduced silver methods in the following aspects. First, the sHRP method does not suffer from the capriciousness which is characteristic of the reduced silver methods. Within the system examined here and under the conditions used with M1 and Sr HRP, injection of the enzyme always resulted in numerous sHRP-labeled neurons afferent to the injection site. Second, the time involved in processing large tissue blocks is considerably shorter (as little as 3-4 days from injection to staining for HRP vs. several weeks or months for Golgi). Third, the structural detail provided by the sHRP method often equals that produced by the Golgi methods. Furthermore, this resolution will certainly be improved even further by additional refinements in the procedure. Fourth, with the sHRP method, information is obtained not only about the dendritic and spinous configuration of a neuron but also about its axonal projection. Finally, by preselecting the site and size of the injection, it is possible to selectively label neurons within a particular cytoarchitectonic population. The Golgi method, on the other hand, often provides a more distinct picture of the cellular processes than does the sHRP method. HRP-stained processes appear finer and are often more difficult to follow. Secondly, with the Golgi method one can visualize not only long axon type l neurons but also the Golgi type II neurons which possess short axons. As presented here this sHRP method is of use for studying neurons with long axons, and therein lies its advantage. Preliminary data and Fig. I, however, indicate that this method may also hold promise for sHRP staining of both type I and type 11 neurons within the periphery of the injection site. Finally, the Golgi method has been applied with success to numerous neuronal systems, while the universality of the sHRP method remains to be established. The intriguing observation that a protein which is taken up by the distal portions of the axon and transported somatopetally to the perikaryon can then be transported somatofugally to the distal-most portions of the dendrites confirms the recent observations of Kreutzberg and his colleagues. They have elegantly shown tha~, following intrasomatic injection of precursor amino acids, endogenous proteins are rapidly distributed throughout the dendritic trees of neurons ~e-54. The rate of intradendritic transport, which is calculated to be from 50 to 200/~m/min44, ~2, is too rapid to be accounted for on the basis of diffusion alone and, since the dendritic transport is blocked by colchicine, the modus operandi probably involves the microtubule system of the ce1144,,53. The use of this solid HRP method may prove useful in studies of dendritoplasmic transport. Since the osmicated DAB product is electron opaque, the movement of the membrane-bound and soluble HRP can theoretically be followed on the EM level from its point of axonal uptake, along the axon, through the perikaryon and throughout the dendritic processes thereby possibly allowing assessment of the relationship of the in transit HRP to the neurotubular system which is thought to be involved in axonal and dendritic transport21,a 1.

30 NOTE ADDED IN PROOF Since s u b m i t t i n g this manuscript, A d a m s a n d W a r r ~ have reported s H R P labeling of n e u r o n s within 3 m m of injection site in 10 o/ /o of kittens 24 h after application o f SgVI H R P to the transected acoustic striae. ACKNOWLEDGEMENTS I a m grateful to Professor Dr. R. Hassler, head of the N e u r o b i o l o g y D e p a r t ment, a n d to the Max-Planck-Gesellschaft for their generous support. I also wish to express my gratitude to Dr. W. Hirschberger for his expert guidance and assistance d u r i n g the p r e p a r a t i o n of the Golgi material, a n d to Dr. J. Christ, in whose laboratory this work was carried out. I a m also t h a n k f u l to Dr. L. Heimer (Virginia) for his helpful c o m m e n t s in preparing the manuscript. A b b r e v i a t i o n s used: D A B - 3, 3 ' - d i a m i n o b e n z i d i n e tetrahydrochloride; D M S O - dimethylsulfoxide; H R P - horseradish peroxidase; d H R P - diffuse H R P ; d g H R P diffuse-granular H R P ; g H R P - g r a n u l a r H R P ; s H R P - solid H R P ; MI H R P Miles H R P ; Sgll H R P - Sigma type I1 H R P ; SgVI H R P - Sigma type VI H R P ;

-

Sr H R P

-

Serva H R P ; PBS - phosphate buffered saline; P G - p a r a f o r m a l d e h y d e -

glutaraldehyde.

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