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Quantification of branched neuronal projections labelled by retrograde fluorescent tracing. A study of olivo-cerebellar projections
Journal of Neuroscience Methods, 16 (1986) 175-189
175
Elsevier
N S M 00572
Quantification of branched neuronal projections labelled by retrograde fluorescent tracing. A study of olivo-cerebellar projections I.N.C. Lawes and J.N. Payne Department of Anatomy and Cell Biology, The University of Sheffield, Sheffield ( U.K.) (Received October 16th, 1985) (Revised January 26th, 1986) (Accepted February 12th, 1986)
Key words: collateral - fluorescent tracer - double labelling - olivocerebellar projection - cell count A method for estimating the number of branched and unbranched neurones projecting from a nucleus to two target sites is presented, based on the retrograde transport of fluorescent tracers. The method initially involves stereological corrections for the size of cytoplasm and nuclei respectively labelled by the two tracers. A second correction is applied to account for doubly labelled cells whose cytoplasm, but not nuclei, are in the plane of section. Finally, the detection rates of the two tracers are determined and appropriate corrections are applied. The projection from the medial accessory olive to the cerebellar vermis was studied using true blue and diamidino yellow to illustrate the method. Application of the method increased the number of branched neurones detected by 18.5%. Of the total increase, 48.4% was due to the correction for size, 9.2% to the correction for doubly labelled cells with nuclei outside the plane of the section and 42.4% to the correction for detection rates. There was no significant masking of one tracer by another, but true blue enhanced the fluorescence of diamidino yellow.
Introduction
The introduction of retrogradely transported fluorescent tracers (Kristensson et al., 1971; Kuypers et al., 1977; Kuypers et al., 1980; Sawchenko and Swanson, 1981; Swanson, 1983) has had a considerable impact on the mapping of neural pathways. One reason for this is that, for the first time, it has become anatomically possible to identify individual neurones which have multiple projections; thus when a different tracer is injected into each of two projection sites such neurones are retrogradely labelled with both (Van der Kooy et al., 1978). There are, however, problems associated with obtaining a numerical estimate of the proportion of branched neurones: (a) When
two
tracers
are used,
one
of which
labels
cytoplasm
and
one
the
Correspondence: I.N.C. Lawes, Department of A n a t o m y and Cell Biology, The University of Sheffield, Sheffield S10 2TN, U.K.
0165-0270/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
176
nucleus, then neurones labelled with the cytoplasmic tracer will be counted more often because the cytoplasm of a cell appears in more sections than the nucleus (DeHoff and Rhines, 1961). (b) Tracers have different diffusion rates, many workers considering that some tracers may diffuse further than others at the injection site and therefore label more neurones retrogradely. (c) It is possible that there are different affinities for uptake of tracers, and that the tracers, once taken up, are retrogradely transported with different efficiencies (of both rate and quantity). (d) Once transported to the cell body or nucleus, the intensity of fluorescence of the tracers may differ, leading to different detection rates. (e) Tracers may interact. Masking, leading to underestimation of the number of branched neurones, has been reported (Ad6r et al., 1980: Alheid et al., 1984b). By contrast, the possibility that fluorochromes may mutually excite each other has been termed scintillation (BjOrklund and Skagerberg, 1979). Both phenomena could alter estimates of the number of branched neurones. (f) Some tracers rapidly leak out of retrogradely labelled neurones (Aschoff and Hollander, 1982, Kuypers et al., 1979) into neighbouring cells, giving spuriously high estimates. This can be circumvented by reducing survival times so that neighbouring glial cells are not labelled (Bentivoglio et al., 1980) and by using tracers which leak from neurones only very slowly (Keizer et ai.~ 1983). The most comprehensive method of quantification (Alheid et al., 1984a) depends on the use of one tracer injected into 3 groups of animals. Injections are made into one of two sites in one group, into the other site in a second group, and into both sites in the final group. The number of neurones seen is then compared in the 3 corresponding groups of animals. There are several problems with this approach, however: first the variance in the number of neurones labelled is large so a large number of animals is required; secondly, if branched neurones are different in size from unbranched neurones they will be incorrectly estimated since they cannot be distinguished from unbranched neurones; finally, tile algebraic approach is applicable only if the detection rate is 100% (the derivation given by Alheid et al. for a detection rate of 80% contains an error which cannot be avoided unless the detection rates are known, in which case there would be no problem with the use of two interacting tracers either, see Discussion). In view of the difficulties which weaken the algebraic approach using one tracer, the numerical aspects involved in using two tracers were re-examined.
Materials and Methods
Under general anaesthesia (ketamine 50 m g / k g i.p. and pentobarbitone 20 m g / k g i.p.) male rats weighing 280-320 g were positioned in a David Kopf stereotaxic frame. Using a glass micropipette (tip diameter 25-50 ttm), linked hydraulically to a Hamilton 7102 microsyringe, a 50 nl suspension of 2% diamidino yellow (DY), 2% true blue (TB) or both tracers (2% each) was injected into the
177 vermis of the cerebellar cortex. Postoperative analgesia was provided by infiltrating the skin of the scalp with 0.5% bupivicaine. After 4 days survival the rats were deeply anaesthetized and perfused through the aorta, first with 0.9% heparinized saline and then (for 15 min) with 10% phosphatebuffered formalin followed by a further 15 min with the same fixative in 15% sucrose. The brains were left in 30% phosphate-buffered sucrose until they sank, then 30/~m transverse or horizontal sections of the medulla and cerebellum were cut in a cryostat. The sections were air-dried, mounted in DPX and viewed with a Leitz Orthoplan fluorescence microscope using mirror-filter A (wide band UV excitation). Profiles of labelled neurones were identified as true blue, diamidino yellow or doubly labelled. The number of each type was counted in every sixth section of the labelled part of the inferior olive. In one animal injected with both tracers sagittal sections were used to determine the rostrocaudal diameters of cells and cell nuclei. Except in this case, results are presented as means (and standard errors) from several animals. The difference between the fluorescence intensity of diamidino yellow labelled cell nuclei excited by ultra-violet (340-380 nm) or short wavelength visible light (400-425 nm) in singly labelled or doubly labelled neurones was measured using a Leitz MPV2 Compact photometer. To exclude the effects of true blue emission an additional barrier filter was used. This was opaque to wavelengths shorter than 490 nm, blocking true blue emission but passing the longer wavelength emissions of diamidino yellow (J.N. Payne, unpublished observations). The photometric measuring diaphragm was adjusted so that measurements were confined to the nucleus or cytoplasm.
Results Derivation of method The effect of cell size on the counts. The effect of size on the number of cells seen in a section is well known (DeHoff and Rhines, 1961). An illustration of the effect as applied to doubly labelled neurones is given in Fig. 1, where the larger outlines represent cytoplasm labelled with one tracer and the smaller outlines represent nuclei labelled with another. As can be seen from this edge-on view of a section, the cytoplasmic tracer will be seen a greater number of times than the nuclear tracer. The centre of each cell is a point and the correction for the number of central points in a section of thickness (t) is given by Abercrombie (1946): N = P. t/(t + L)
(1)
where L is the mean length of cytoplasm or nucleus perpendicular to the plane of section, N is the true number of centres per section and P is the number of profiles counted in each section. There is a possibility that neurones which branch may be of a different size from those which do not, as illustrated by the medium spiny and large aspiny neurones of
178
0© ©© Lc Fig. 1. Edge-on view of a section passing through a population of cells. The cytoplasm of 8 cells is visible in the section, but only 7 nuclei appear. This discrepancy can be corrected if the mean length perpendicular to the section is known (L n for the nucleus, L~ for the cytoplasm). the striatum (Carpenter, 1984; Henderson, 1981; Preston et al., 1980). In this case doubly labelled cells will have a different probability of being counted from singly labelled cells. It is therefore necessary to test for difference in size between branched and unbranched neurones. This requires that the branched neurones are individually identified, which can only be achieved when they are doubly labelled. If a single tracer algebraic technique is used the identity of branched neurones is concealed and there is no possibility of correcting for differences in size. The number of centres ( N h) derived from cytoplasmic profiles (Pb) labelled with true blue is given by: Nb = Pb" t / ( t + Lbc )
(2)
where Lbc is the length of blue labelled cytoplasm perpendicular to the section. Similarly, the number of centres (Nv) derived from nuclear profiles (Py) labelled with diamidino yellow is: Nv = Py . t / ( t + Lyn)
(3)
where Ly n is the length of nuclei of singly labelled neurones. Finally, Nd is the number of doubly labelled cell centres derived from doubly labelled profiles (Pd): Ud = Pd" t / ( t + Ld,)
(4)
where La, is the length of nuclei of doubly labelled neurones. Obviously, doubly labelled cells can only be identified as such when their nuclei are in the section, hence the use of nuclear length in the correction. Misidentified doubly labelled cells. Some of the profiles apparently labelled only with true blue have their nuclei out of the plane of the section. A proportion of these would actually be doubly labelled cells. The correction for these misidentified cells is as follows.
179 The number of doubly labelled cell centres (No) is obtained by applying Abercrombie's correction to the number of doubly labelled nuclear profiles (Pa). If the cytoplasmic profiles of these cell centres could also have been correctly identified, then inverting Abercrombie's correction gives the number of expected cytoplasmic profiles (Pc): Pe = N d .(Ldc + t ) / t
(5)
where Lo¢ is the cytoplasmic length, perpendicular to the plane of the section, of doubly labelled neurones. The observed number of doubly labelled nuclear profiles (P0) is~ subtracted from this expected number of cytoplasmic profiles (P~). This difference represents the number of cells whose cytoplasmic profiles were labelled blue, but had their nuclei (labelled with diamidino yellow) out of the plane of the section. These profiles are incorrectly attributed to true blue labelled cells. They increase the number of such cells by N m, where: Um = ( ee - Pd )" t / ( tdc + t )
(6)
It follows that, if there are N b centres attributed to true blue labelled neurones, then only N b - N m centres actually belong to cells labelled exclusively with true blue, since N m centres are in fact doubly labelled. Detection rates. A difficulty in the use of two tracers is that they may interact with each other, one masking the other (Alheid et al., 1984a) or possibly increasing its detection rate (Lenn et al., 1983). It is important therefore to estimate the detection rates for the chosen tracers; first when both dyes are injected into the same site, then when they are injected separately in different groups of animals. If there are N neurones projecting to a site which has been injected with a mixture of two tracers, ([N b - N m ] labelled with true blue alone, Ny labelled with diamidino yellow alone and No labelled with both, i.e. double labelled), then the detection rates for true blue (Rb) and diamidino yellow (Ry) are given by: R b = ( [ N b - Nm] + N d ) / N
(V)
Ry = (Ny + N d ) / N
(8)
The detection rate for doubly labelled neurones will be the conjoint detection rate of both tracers, given by: R b .Ry = Nd/N
(9)
By substitution from Eqn. 9, the detection rates become: Rb = N d / ( Ny + gd)
(10)
R y = N d / ( N b - N m + Nd)
(11)
Detection rates and branched neurones
Once the detection rates have been determined from injections of mixtures into the same site, they can be used to correct the number of branched neurones counted after single injections into two sites. After the number of such profiles has been
180 corrected for both size and misidentification of doubly labelled cells (as described previously), the number of cell centres (C b for true blue, Cv for diamidino yellow and Cd for doubly labelled neurones) is corrected for detection rates as follows. Ch = (Nh -- N m ) / R h
(12)
(\. = N,,/R,
(13)
Cd = Nd/( R h • Ry )
(14)
A short computer program incorporating the corrections is available from the authors.
Experimental determination of detection rates Data obtained from labelling cells of the olivocerebellar pathway with a mixture of tracers injected at one site will now be presented. The various corrections will be applied in sequence in Table II. Correction for size. The rostrocaudal lengths of nuclei and cytoplasm for singly and doubly labelled cells were determined and found to be the same in neurones of this particular pathway (Table I). The relative lengths of nuclei and cytoplasm obtained indicate that cytoplasmic tracers will label 16% more profiles (in the 30/~m thick sections used) than nuclear tracers. The number of profiles labelled with each tracer was counted (column 1, Table II). The mean number of cell centres in a section (column 2) was derived from the number of profiles courted by applying Eqn. 2 to true blue labelled neurones and Eqn. 3 to diamidino yellow labelled and doubly labelled neurones. Correction for misidentified doubly labelled cells. After injecting a mixture of dyes, the mean number of nuclear profiles observed was 207 (Table II). The number of doubly labelled cell centres, obtained from applying Abercrombie's correction to the number of doubly labelled nuclear profiles, was 145 (Eqn. 4). If the cytoplasmic profiles of these 145 cell centres could also have been correctly identified, then inverting Abercrombie's correction gives the number of expected cytoplasmic profiles as 241 (Eqn. 5). Consequently, 34 (241-207) cytoplasmic profiles were labelled blue, but their nuclei were out of the plane of the section and were labelled with diamidino yellow. This corresponds to 20 missed cell centres (Eqn. 6) which belonged to doubly labelled cells but were apparently labelled only by true blue. It follows that 19 ( 3 9 - 20) centres belonged to cells singly labelled with true blue (column 3, Table II).
TABLE 1 THE MEAN (4- S.E.M.) ROSTROCAUDALLENGTH IN /~m OF FLUORESCENT LABELLED NEURONES IN THE MEDIAL ACCESSORYOLIVE OF ONE RAT (n = 14) Singly labelled Doubly labelled
Nuclei
Cytoplasm
13.1 _+0.2 12.84-0.5
20.34-0.4 20.3 + 0.7
181 TABLE II NUMBERS OF LABELLED PROFILES AND CELL CENTRES The numbers are the totals (+_ S.E.M.) obtained from both sides of every sixth section through the medial accessory olive, averaged across animals. Number of animals Mixture injection TB DY DL Single injection TB DY
Profiles
Cell centres (corrected for size)
Cell centres (corrected for misssed DL)
65.3+ 9.0 2.7_+ 0.6 207.2 _+22.7
39.2_+ 5.4 1.9_+ 0.4 1'44.5+ 15.9
19.0_+ 3.7 1.9_+ 0.4 144.5 _+15.9
304.2 _+53.3 180.0 _+32.9
182.9 _+31.8 125.7 _+23.0
6
8 7
D e t e c t i o n rates. Eqns. 10 and 11 were applied to the data in Table II. The detection rate for true blue (R b) was 0.986 + 0.003, and for diamidino yellow ( R y ) it was 0.886 + 0.014. I n t e r a c t i o n b e t w e e n tracers
The most frequent observation in the literature is that the presence of one tracer m a y mask another (Ad6r et al., 1980; De Olmos and Heimer, 1980; Swanson, 1983; Alheid et al.,, 1984). In the present experiments, after injections of a mixture of two tracers, the total n u m b e r of profiles labelled with true blue was 272 (207 + 65), and with diamidino yellow, 210 (207 + 3). For comparison, tracers were injected singly in the same site in different animals. After these single tracer injections (Table II), the mean n u m b e r of profiles labelled with true blue was 304, and with diamidino yellow, 180. Thus more profiles were labelled with true blue and fewer with diamidino yellow. This raises the possibility that diamidino yellow in the nuclei of double labelled cells might have absorbed light emitted by true blue in the surrounding cytoplasm, i.e. that scintillation was occurring (Bj0rklund and Skagerberg, 1979). True blue emits light of a wavelength capable of exciting diamidino yellow, so that true blue in the cytoplasm may increase the excitation of diamidino yellow in the nucleus, thereby increasing the detection rates. This hypothesis was tested by exciting singly and doubly labelled cells, first with ultra-violet light of 340-380 nm (which excites both true blue and diamidino yellow), then with short wavelength visible light of 400-425 nm (which excites mainly diamidino yellow). A second group of cells was excited first with visible light then with ultra-violet light, to control for order effects. The background or cytoplasmic levels were subtracted from each reading then the ratio of the intensity emitted with ultra-violet excitation to that with visible light excitation was calculated. N o significant differences were found in relation to the order in which visible and ultra-violet light were used, so the two groups (visible light first and ultra-violet light first) were combined.
182
2.0
1.6
~
:6 ~ 1.2
-
~ 0.8 n 0,4
0.0
.
.
.
DL
. DY
. DY
DL
(a)
(b)
Fig. 2. Photometric m e a s u r e m e n t s o b t a i n e d from the nuclei of d o u b l y labelled ( D E ) and singly labelled ( D Y ) neurones, a: c y t o p l a s m i c m e a s u r e m e n t subtracted from nuclear m e a s u r e m e n t , b: b a c k g r o u n d m e a s u r e m e n t s u b t r a c t e d from nuclear measurement. O p e n bars: ultra-violet excitation. Filled bars: short w a v e l e n g t h visible excitation. A r b i t r a r y units.
2.4--
e.D
•o ¢1
2.0-
o~
1.6
¢' i,.
m
•
•
• $
"~
L+
& 4',,
1.2-
"," II
0 ,+,., "~
0
"=
--
0.8
0.4
a:
0.0
%
1
o
DL
DY (a)
DL
DY (b)
Fig. 3. Ratios of photometric measurements obtained from nuclei with ultra violet and visible light excitation, a: cytoplasmic measurement subtracted from nuclear measurement, b: background measurement subtracted from nuclear measurement. DL, doubly labelled neurones; DY. singly labelled nuclei.
183 In doubly labelled neurones the quantity of light emitted from nuclei relative to cytoplasm with ultra-violet excitation (Fig. 2) was greater than with short wavelength visible excitation. For diamidino yellow nuclei the opposite was true. The median ratio of emitted light with ultra-violet excitation to emitted light with short wavelength visible excitation was 75% greater for doubly labelled than for diamidino yellow neurones (Fig. 3). The ratios were subjected to a M a n n - W h i t n e y U-test, which showed a highly significant difference between the doubly labelled and diamidino yellow neurones ( U = 5, P < 0.001). When the light emitted from the nuclei was measured relative to background levels, a similar difference was found (U = 3, P < 0.001). Thus more light was emitted from diamidino yellow in the nuclei of neurones which contained true blue in the surrounding cytoplasm when they were excited by ultra-violet light than when short wavelength visible light was used. This was despite the use of a filter to block the light emitted by true blue itself, and was still true even if the emission from the cytoplasm was first subtracted. This may explain why, when the number of true blue labelled profiles decreased after injections of a mixture of two tracers, the number of diamidino yellow profiles increased. Fortunately the difference in the number of profiles counted after mixture and single tracer injections in our experiments did not reach statistically significant levels (X 2 = 3.76, df = 1), suggesting that the detection rates were not substantially altered by the presence of a second tracer. Thus, although interactions of fluorescent intensity were occurring, the initial levels were sufficiently above visual threshold for this interaction not to affect the counts significantly. Two sets of detection rates must be used if significant interactions are found, one set applying to doubly labelled neurones where interactions occur, and the second set to singly labelled neurones.
Experimental determination of proportion of branched neurones in the olivocerebellar pathway As an illustration of the present method, 2% true blue was injected into the posterior lobe of the cerebellar vermis and 2% diamidino yellow was injected into the anterior lobe. The posterior injections were in lobules VI and VII and the anterior injections were in lobules IV and V (Fig. 4). Only injections which had their maximum extent in the same sagittal plane without overlap of the two tracers were accepted (Payne et al., 1985). Data obtained from the medial accessory olive gave the numbers of branched and unbranched neurones presented in Table III. Three corrections were used. The first, from row 1 to row 2, corrects for the difference in size of nuclei and cytoplasm and reduces the difference between the percentages of true blue and diamidino yellow labelled neurones. The second, from row 2 to row 3, corrects for centres belonging to doubly labelled cells being counted as true blue labelled cytoplasmic profiles. The third, rows 3-4, corrects for the different detection rates of the two tracers. Since these injections were in a similar site to that used earlier in mixture injections, the same detection rates were assumed to apply. The proportion of branched neurones, underestimated by raw profile counts, was increased by 18.5%. Of this total increase,
184 Diamidlno Yellow
ANTERIOR
POSTERIOR
Fig. 4. A section through the cerebellar vermis demonstrating the extent of the injection sites in an animal used to determine numbers of branched neurones.
48.4% was d u e to the c o r r e c t i o n for size. A f u r t h e r 9.2% o f the i n c r e a s e was d u e to the c o r r e c t i o n for d o u b l y l a b e l l e d n e u r o n e s m i s i d e n t i f i e d as true b l u e l a b e l l e d cells b e c a u s e their nuclei w e r e n o t in the p l a n e of the section, a n d 42.4% was d u e to the d i f f e r e n c e in d e t e c t i o n rates of true b l u e a n d d i a m i d i n o yellow. Variability of numbers detected. It is e v i d e n t f r o m the tables that there is c o n s i d e r a b l e v a r i a t i o n in the n u m b e r of p r o f i l e s l a b e l l e d w i t h a p a r t i c u l a r tracer. A f t e r i n j e c t i o n s o f m i x t u r e s , the c o e f f i c i e n t of v a r i a t i o n in the n u m b e r of p r o f i l e s was 27.7% for all true b l u e cells a n d 26.3% for all d i a m i d i n o y e l l o w cells, w h e r e a s the
TABLE III MEAN NUMBERS (AND S.E.M.) OF BRANCHED NEURONES PROJECTING TO TWO SITES IN THE CEREBELLAR VERMIS, SUMMED OVER 6 SECTIONS THEN AVERAGED ACROSS 5 ANIMALS (n = 5) True blue Profiles Cell centres Correction for: Size Missed DL Detection rate
Doubly labelled
Diamidino yellow
No.
%
No.
No.
%
164.8 (49.0)
58.8
27.8 (7.7)
9.9
8"/.6 (10.9)
31.3
98.9 (29.4) 96.2 (29.1) 97.5 (29.5)
55.1 54.4 51.7
19.4 (5.4) 19.4 (5.4) 22.2 (6.1)
10.8 11.0 11.8
61.1 ("/.6) 61.1 ('/.6) 69.0 (8.6)
34.1 34.6 36.6
%
185 coefficients for the detection rates were 0.8% and 3.7% respectively. Clearly, the detection rate is a robust measure whereas the absolute number of profiles is very variable. The most likely explanation is variation in injection size between animals.
Discussion The use of two retrogradely transported fluorescent tracers was one of the first anatomical techniques to identify branched neurones qualitatively (Van der Kooy et al., 1978). The present paper suggests an analytical procedure to quantify the proportion of branched neurones in a population of cells projecting to two injection sites. The corrections which have to be applied to the number of profiles counted in order to convert them into relative numbers of neurones involve 3 stages. The first two stages depend on knowing the size of branched and unbranched neurones and their nuclei. Clearly, nuclear and cytoplasmic profiles differ in size, and unless corrections for this are employed, errors will be made. It is interesting to note that, in their criticism of the double labelling technique, Alheid et al. (1984b) found no significant difference in the number of cells labelled with nuclear yellow and granular blue after unilateral injections in 26/~m thick sections despite the fact that one tracer labels nuclei while the other labels cytoplasm. This suggests that there were differences in the detection rates of the tracers they used, or that leakage had occurred. Branched neurones may differ in size from unbranched neurones so there will be an error of estimation if the two are not distinguished. This is an unavoidable error if the single tracer algebraic technique of Alheid et al. (1984a, 1984b) is used. It is therefore essential that branched neurones are identified individually to assess their relative size, and this requires double labelling. In the method presented here, stage I is a conventional correction for different sizes of the cellular compartments labelled by two tracers. In the present experiments, this correction accounted for 48.4% of the total increase in the relative number of branched neurones. Stage II reclassifies a proportion of cytoplasmically labelled profiles as being double labelled but with their nuclei out of the plane of the section. The final correction, stage III, takes into account the different detection rates of the two tracers. This accounted for 42.4% of the total increase. It is sometimes assumed that true blue is detected more frequently than diamidino yellow because it diffuses further at the injection site. After the two stereological corrections of stage I and II, the detection rates of the two tracers were within 10% of each other (99% and 89% respectively). Other factors contributing to differences in detection rate, besides diffusion, are: the placement, volume and concentration of tracers; the affinity for uptake of the tracers; the efficiency and rate of retrograde transport; the fluorescent intensity of the tracers; leaking from the cells; masking and scintillation. One effect of injection site placement was obviated by the selection of only those cases in which the maximum extent of the injections was in the same sagittal plane
186 and there was no overlap between them. The possible effect of different sites on detection rates, such as unequal distances to travel, was avoided by determining the rates after injections in the same place as in the experimental groups. Of the other factors mentioned, only masking and scintillation were specifically investigated in the present experiments. The nuclei of singly labelled diamidino yellow neurones emitted more light when short wavelength visible excitation was used than when they were excited by ultra-violet light, as predicted from the absorption spectrum of diamidino yellow. However, photometric measurements indicated that a diamidino yellow labelled nucleus emitted more light with ultra-violet excitation if there was true blue in the surrounding cytoplasm. This strongly suggests that true blue emissions were secondarily exciting diamidino yellow. The emission spectrum of true blue is coextensive with the absorption spectrum of diamidino yellow, so that if cells were doubly labelled their nuclei fluoresced more intensely than if they were singly labelled. However, although the number of profiles labelled by diamidino yellow was 16.6% greater after injections of mixtures than after single tracer injections, the increase was not substantial enough to affect the detection rate of this tracer significantly. This implies that the intensity of fluorescence of singly labelled nuclei was already above visual threshold, or that the variation between animals was too great to reveal the effects of scintillation and masking. The tendency of diamidino yellow to mask true blue (by absorbing its emissions), indicated by a 10.4% reduction, was not statistically significant either. A possible explanation of why Alheid et al. observed so much masking is that there was extensive leakage from transporting cells to their neighbours, giving them spuriously high counts. If, however, there is a significant interaction between tracers, it is possible to allow for this by incorporating the altered detection rates in the equations presented here. The absolute number of neurones labelled by retrograde tracers is notoriously variable. In the present experiments, coefficients of variation of 25% or more were obtained, in line with the results of other investigators (Alheid et al., 1984b; Cavada et al., 1984; Huisman et al., 1983; IUert et al., 1982; Luskin and Price, 1982; Room et al., 1981; Sawchenko and Swanson, 1981). Given this variation, any technique which relies on absolute counts requires large numbers of animals to obtain reliable results. This drawback applies to the algebraic method of Alheid et al. (1984a), where, to detect the small proportion of branched neurones obtained here, one would require approximately 20 animals for each comparison. By contrast, the coefficients of variation for the detection rates of the two tracers were less than 4% across animals. It is inherently more satisfactory to use each animal as its own control whenever possible and the double labelling technique permits exactly this. At present, therefore, quantification gives the relative, but not the absolute, numbers of branched neurones in a population. In addition to the practical problems involved in implementing the algebraic approach (Alheid et al., 1984a, 1984b) discussed above, there is a theoretical limitation to their technique, making it applicable only if the detection rate is 100%. The derivation given by Alheid et al. for a detection rate of 80% contains an error which cannot be avoided unless the detection rates are known. The error in their derivation is as follows: K a is the number of profiles labelled by an injection of a
187 single tracer into one of two sites, K b the number after injection into the other site and Kab is the number after injection of the same tracer into both. The number of cells projecting to the two sites is A and B and to both sites, AB. The equations they gave were: K a = 80%A + 80%AB
(a)
80%AB + 80%B
(b)
Kb=
Kab = 8 0 % A + 8 0 % A B + 8 0 % B
(c)
From (a-c) they derived the proportion ( P ) of branched neurones: P = AB/(A
+ AB + B)
(d)
The errors occurs in Eqn. c. Of the branched neurones, 80% would be labelled from site A, but a further 80% of the neurones unlabelled from site A (20%) would be labelled from site B (i.e. 16%), making 96% (80% + 16%) altogether. Thus Eqn. c should be: Kah = 8 0 % A + 9 6 % A B + 8 0 % B
(e)
Using this corrected equation, the number of branched neurones is given by Ka + K b - Kab, o r ( 8 0 + 80 -- 96)%AB. Thus the proportion of branched neurones becomes: P = 6 4 % A B / ( 8 0 % A + 96%AB + 80%B)
(f)
It can be seen that the proportion of branched neurones cannot be calculated until the detection rate is known. This is a serious constraint for the algebraic approach because, of source, there is no way of knowing the detection rate for a single tracer technique. It is only when double labelling is employed that detection rates (including the effect of interaction on these) can be measured. In applying a quantitative method, it is important not to use it outside its limitations, and to bear in mind the difference between relative and absolute numbers. This paper provides a quantitative analysis of the proportion of branched neurones, correcting for stereological errors and differences in detection rate of the fluorescent tracers. It is perhaps reassuring that after the corrections employed, the final proportion of branched neurones increased by a relatively small amount. For neurones of different size, counted in sections of different thickness, the changes resulting from the present corrections may be much larger. If, therefore, it is of importance to have accurate results, then the present corrections are recommended. On the other hand, the present results indicate that raw profile counts may be sufficient for many purposes. Finally, the location of branching neurones can be determined only by using a double labelling technique. In some cases branched neurones occupy a distinct location (Payne et al., 1985). By providing quantitative information on both the location and the proportion of neurones which branch, the double retrograde tracer technique contributes greatly to the unravelling of neuronal circuitry.
188
Acknowledgements The authors would like to thank C.J. Hill and K. Crawford for their technical assistance. We would also like to thank S.M. Wharton for contributing to the counts in Table III.
References Abercrombie, M. (1946) Estimation of nuclear populations from microtome sections, Anat. Rec.. 94: 239-247. Ad6r, J-P., Room, P., Postema, F. and Korf, J. (1980) Bilaterally diverging axon collaterals and contralateral projections from rat locus coeruleus neurons, demonstrated by fluorescent retrograde double labeling and norepinephrine metabolism, J. Neural Transm., 49: 207-218. Alheid, G.F., Carlsen, J. and Heimer, L. (1984a) An algebraic approach to the detection of multiple markers in complex neuronal systems, J. Neurosci. Meth., 10: 71-77. Alheid, G.F., Carlsen, J., de OImos, J. and Heimer, L. (1984b) Quantitative determination of collateral anterior olfactory nucleus projections using a fluorescent tracer with an algebraic solution to the problem of double retrograde labeling, Brain Res.. 292: 17-22. Aschoff, A. and Hollander, H. (1982) Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). I. A parametric study in the central visual system of the adult rat, J. Neurosci. Meth., 6: 179-197. Bentivoglio, M., Kuypers, H.G.J.M. and Catsman-Berrevoets, C.E. (1980) Retrograde neuronal labeling by means of bisbenzimide and nuclear yellow (Hoechst 769121). Measures to prevent diffusion of the tracers out of retrogradely labeled neurons, Neurosci. Lett., 18: 19-24. Bjorklund, A. and Skagerberg, G. (1979) Simultaneous use of retrograde fluorescent tracers and fluorescence histochemistry for convenient and precise mapping of monoaminergic projections and collateral arrangements in the C.N.S., J. Neurosci. Meth., 1: 261-277. Carpenter, M.B. (1984) Interconnections between the corpus striatum and brain stem nuclei. In J.S. McKenzie, R.E. Kemm and L.N. Wilcock (Eds.), The Basal Ganglia, Structure and Function, Plenum Press, New York. Cavada, C., Huisman, A.M. and Kuypers, H.G.J.M. (1984) Retrograde double labeling of neurons: the combined use of horseradish peroxidase and diamidino yellow dihydrochloride (DY. 2HC1) compared with true blue and D1Y.2HCI in rat descending pathways, Brain Res., 308: 123-136. DeHoff, R.T. and Rhines, F.N. (1961) Determination of the number of particles per unit volume from measurements made on random plane sections: the general cylinder and ellipsoid, Trans. Am. Inst. Mineral Met. Eng., 221: 975-982. De Olmos, J. and Heimer, L. (1980) Double and triple labeling of neurons with fluorescent substances: the study of collateral pathways in the ascending raphe system, Neurosci. Left., 19: 7-12. Henderson, Z. (1981) Ultrastructure and acetylcholinesterase content of neurones forming connections between the striatum and substantia nigra of rat, J. Comp. Neurol., 197: 185-196. Huisman, A.M., Kuypers, H.G.J.M., Conde, F. and Keizer, K. (1983) Collaterals of rubrospinal neurons to the cerebellum in the rat. A retrograde fluorescent double labeling study, Brain Res., 264: 181-196. Illert. M., Fritz, N., Aschoff, A. and Hollander, H. (1982) Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). II. A parametric study in the peripheral motor system of the cat, J. Neurosci. Meth., 6: 199-218. Keizer, K., Kuypers, H.G.J.M., Huisman, A.M and Dann, O. (1983) Diamidino yellow dihydrochloride (DY. 2HCI); a new fluorescent retrograde neuronal tracer which migrates only very slowly out of the cell, Exp. Brain Res., 51: 179-191. Kristensson, K., Olsson, Y. and Sjostrand, J. (1971) Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain R.es., 32: 399-406. Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Padt, R.E. (1977) Retrograde axonal transport of fluorescent substances in the rat's forebrain, Neurosci. Lett., 6: 127-135.
189 Kuypers, H.G.J.M., Bentivoglio, M., Van der Kooy, D. and Catsman-Berrevoets, C.E. (1979) Retrograde transport of bisbenzimide and propidium iodide through axons to their parent cell bodies, Neurosci. Lett., 12: 1-7. Kuypers, H.G.J.M., Bentivoglio, M., Catsman-Berrevoets, C.E. and Bharos, A.T. (1980) 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: 383-392. Lenn, N.J., Wong, V. and Hamill, G.S. (1983) Left-right pairing at the crest synapses of rat interpeduncular nucleus, Neuroscience, 9: 383-389. Luskin, M.B. and Price, J.L. (1982) The distribution of axon collaterals from the olfactory bulb and the nucleus of the horizontal limb of the diagonal band to the olfactory cortex, demonstrated by double retrograde labeling techniques, J. Comp. Neurol., 209: 249-263. Payne, J.N., Wharton, S.M. and Lawes, I.N.C. (1985) Quantitative analysis of the topographical organization of the olivo-cerebellar projections in the rat, Neuroscience, 15: 403-415. Preston, R.J., Bishop, G.A. and Kitai, S.T. (1980) Medium spiny neuron projection from the rat striatum: an intracellular horseradish peroxidase study, Brain Res., 183: 253-263. Room, P., Postema, F. and Korf, J. (1981) Divergent axon collaterals of rat locus coeruleus neurons: demonstration by a fluorescent double labeling technique, Brain Res., 221: 219-230. Sawchenko, P.E. and Swanson, L.W. (1981) A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemical techniques, Brain Res., 210: 31-51. Swanson, L.W. (1983) The use of retrogradely transported fluorescent markers in neuroanatomy. In J.L. Barker and J.F. McKelvy (Eds.), Current Methods in Cellular Neurobiology. Volume 1. Anatomical Techniques, Wiley, New York, pp. 219-240. Van der Kooy, D., Kuypers, H.G.J.M. and Catsman-Berrevoets, C.E. (1978) Single mamillary body cells with divergent axon collaterals. Demonstration by a simple, fluorescent retrograde double labeling technique in the rat, Brain Res., 158: 189-196.
175
Elsevier
N S M 00572
Quantification of branched neuronal projections labelled by retrograde fluorescent tracing. A study of olivo-cerebellar projections I.N.C. Lawes and J.N. Payne Department of Anatomy and Cell Biology, The University of Sheffield, Sheffield ( U.K.) (Received October 16th, 1985) (Revised January 26th, 1986) (Accepted February 12th, 1986)
Key words: collateral - fluorescent tracer - double labelling - olivocerebellar projection - cell count A method for estimating the number of branched and unbranched neurones projecting from a nucleus to two target sites is presented, based on the retrograde transport of fluorescent tracers. The method initially involves stereological corrections for the size of cytoplasm and nuclei respectively labelled by the two tracers. A second correction is applied to account for doubly labelled cells whose cytoplasm, but not nuclei, are in the plane of section. Finally, the detection rates of the two tracers are determined and appropriate corrections are applied. The projection from the medial accessory olive to the cerebellar vermis was studied using true blue and diamidino yellow to illustrate the method. Application of the method increased the number of branched neurones detected by 18.5%. Of the total increase, 48.4% was due to the correction for size, 9.2% to the correction for doubly labelled cells with nuclei outside the plane of the section and 42.4% to the correction for detection rates. There was no significant masking of one tracer by another, but true blue enhanced the fluorescence of diamidino yellow.
Introduction
The introduction of retrogradely transported fluorescent tracers (Kristensson et al., 1971; Kuypers et al., 1977; Kuypers et al., 1980; Sawchenko and Swanson, 1981; Swanson, 1983) has had a considerable impact on the mapping of neural pathways. One reason for this is that, for the first time, it has become anatomically possible to identify individual neurones which have multiple projections; thus when a different tracer is injected into each of two projection sites such neurones are retrogradely labelled with both (Van der Kooy et al., 1978). There are, however, problems associated with obtaining a numerical estimate of the proportion of branched neurones: (a) When
two
tracers
are used,
one
of which
labels
cytoplasm
and
one
the
Correspondence: I.N.C. Lawes, Department of A n a t o m y and Cell Biology, The University of Sheffield, Sheffield S10 2TN, U.K.
0165-0270/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
176
nucleus, then neurones labelled with the cytoplasmic tracer will be counted more often because the cytoplasm of a cell appears in more sections than the nucleus (DeHoff and Rhines, 1961). (b) Tracers have different diffusion rates, many workers considering that some tracers may diffuse further than others at the injection site and therefore label more neurones retrogradely. (c) It is possible that there are different affinities for uptake of tracers, and that the tracers, once taken up, are retrogradely transported with different efficiencies (of both rate and quantity). (d) Once transported to the cell body or nucleus, the intensity of fluorescence of the tracers may differ, leading to different detection rates. (e) Tracers may interact. Masking, leading to underestimation of the number of branched neurones, has been reported (Ad6r et al., 1980: Alheid et al., 1984b). By contrast, the possibility that fluorochromes may mutually excite each other has been termed scintillation (BjOrklund and Skagerberg, 1979). Both phenomena could alter estimates of the number of branched neurones. (f) Some tracers rapidly leak out of retrogradely labelled neurones (Aschoff and Hollander, 1982, Kuypers et al., 1979) into neighbouring cells, giving spuriously high estimates. This can be circumvented by reducing survival times so that neighbouring glial cells are not labelled (Bentivoglio et al., 1980) and by using tracers which leak from neurones only very slowly (Keizer et ai.~ 1983). The most comprehensive method of quantification (Alheid et al., 1984a) depends on the use of one tracer injected into 3 groups of animals. Injections are made into one of two sites in one group, into the other site in a second group, and into both sites in the final group. The number of neurones seen is then compared in the 3 corresponding groups of animals. There are several problems with this approach, however: first the variance in the number of neurones labelled is large so a large number of animals is required; secondly, if branched neurones are different in size from unbranched neurones they will be incorrectly estimated since they cannot be distinguished from unbranched neurones; finally, tile algebraic approach is applicable only if the detection rate is 100% (the derivation given by Alheid et al. for a detection rate of 80% contains an error which cannot be avoided unless the detection rates are known, in which case there would be no problem with the use of two interacting tracers either, see Discussion). In view of the difficulties which weaken the algebraic approach using one tracer, the numerical aspects involved in using two tracers were re-examined.
Materials and Methods
Under general anaesthesia (ketamine 50 m g / k g i.p. and pentobarbitone 20 m g / k g i.p.) male rats weighing 280-320 g were positioned in a David Kopf stereotaxic frame. Using a glass micropipette (tip diameter 25-50 ttm), linked hydraulically to a Hamilton 7102 microsyringe, a 50 nl suspension of 2% diamidino yellow (DY), 2% true blue (TB) or both tracers (2% each) was injected into the
177 vermis of the cerebellar cortex. Postoperative analgesia was provided by infiltrating the skin of the scalp with 0.5% bupivicaine. After 4 days survival the rats were deeply anaesthetized and perfused through the aorta, first with 0.9% heparinized saline and then (for 15 min) with 10% phosphatebuffered formalin followed by a further 15 min with the same fixative in 15% sucrose. The brains were left in 30% phosphate-buffered sucrose until they sank, then 30/~m transverse or horizontal sections of the medulla and cerebellum were cut in a cryostat. The sections were air-dried, mounted in DPX and viewed with a Leitz Orthoplan fluorescence microscope using mirror-filter A (wide band UV excitation). Profiles of labelled neurones were identified as true blue, diamidino yellow or doubly labelled. The number of each type was counted in every sixth section of the labelled part of the inferior olive. In one animal injected with both tracers sagittal sections were used to determine the rostrocaudal diameters of cells and cell nuclei. Except in this case, results are presented as means (and standard errors) from several animals. The difference between the fluorescence intensity of diamidino yellow labelled cell nuclei excited by ultra-violet (340-380 nm) or short wavelength visible light (400-425 nm) in singly labelled or doubly labelled neurones was measured using a Leitz MPV2 Compact photometer. To exclude the effects of true blue emission an additional barrier filter was used. This was opaque to wavelengths shorter than 490 nm, blocking true blue emission but passing the longer wavelength emissions of diamidino yellow (J.N. Payne, unpublished observations). The photometric measuring diaphragm was adjusted so that measurements were confined to the nucleus or cytoplasm.
Results Derivation of method The effect of cell size on the counts. The effect of size on the number of cells seen in a section is well known (DeHoff and Rhines, 1961). An illustration of the effect as applied to doubly labelled neurones is given in Fig. 1, where the larger outlines represent cytoplasm labelled with one tracer and the smaller outlines represent nuclei labelled with another. As can be seen from this edge-on view of a section, the cytoplasmic tracer will be seen a greater number of times than the nuclear tracer. The centre of each cell is a point and the correction for the number of central points in a section of thickness (t) is given by Abercrombie (1946): N = P. t/(t + L)
(1)
where L is the mean length of cytoplasm or nucleus perpendicular to the plane of section, N is the true number of centres per section and P is the number of profiles counted in each section. There is a possibility that neurones which branch may be of a different size from those which do not, as illustrated by the medium spiny and large aspiny neurones of
178
0© ©© Lc Fig. 1. Edge-on view of a section passing through a population of cells. The cytoplasm of 8 cells is visible in the section, but only 7 nuclei appear. This discrepancy can be corrected if the mean length perpendicular to the section is known (L n for the nucleus, L~ for the cytoplasm). the striatum (Carpenter, 1984; Henderson, 1981; Preston et al., 1980). In this case doubly labelled cells will have a different probability of being counted from singly labelled cells. It is therefore necessary to test for difference in size between branched and unbranched neurones. This requires that the branched neurones are individually identified, which can only be achieved when they are doubly labelled. If a single tracer algebraic technique is used the identity of branched neurones is concealed and there is no possibility of correcting for differences in size. The number of centres ( N h) derived from cytoplasmic profiles (Pb) labelled with true blue is given by: Nb = Pb" t / ( t + Lbc )
(2)
where Lbc is the length of blue labelled cytoplasm perpendicular to the section. Similarly, the number of centres (Nv) derived from nuclear profiles (Py) labelled with diamidino yellow is: Nv = Py . t / ( t + Lyn)
(3)
where Ly n is the length of nuclei of singly labelled neurones. Finally, Nd is the number of doubly labelled cell centres derived from doubly labelled profiles (Pd): Ud = Pd" t / ( t + Ld,)
(4)
where La, is the length of nuclei of doubly labelled neurones. Obviously, doubly labelled cells can only be identified as such when their nuclei are in the section, hence the use of nuclear length in the correction. Misidentified doubly labelled cells. Some of the profiles apparently labelled only with true blue have their nuclei out of the plane of the section. A proportion of these would actually be doubly labelled cells. The correction for these misidentified cells is as follows.
179 The number of doubly labelled cell centres (No) is obtained by applying Abercrombie's correction to the number of doubly labelled nuclear profiles (Pa). If the cytoplasmic profiles of these cell centres could also have been correctly identified, then inverting Abercrombie's correction gives the number of expected cytoplasmic profiles (Pc): Pe = N d .(Ldc + t ) / t
(5)
where Lo¢ is the cytoplasmic length, perpendicular to the plane of the section, of doubly labelled neurones. The observed number of doubly labelled nuclear profiles (P0) is~ subtracted from this expected number of cytoplasmic profiles (P~). This difference represents the number of cells whose cytoplasmic profiles were labelled blue, but had their nuclei (labelled with diamidino yellow) out of the plane of the section. These profiles are incorrectly attributed to true blue labelled cells. They increase the number of such cells by N m, where: Um = ( ee - Pd )" t / ( tdc + t )
(6)
It follows that, if there are N b centres attributed to true blue labelled neurones, then only N b - N m centres actually belong to cells labelled exclusively with true blue, since N m centres are in fact doubly labelled. Detection rates. A difficulty in the use of two tracers is that they may interact with each other, one masking the other (Alheid et al., 1984a) or possibly increasing its detection rate (Lenn et al., 1983). It is important therefore to estimate the detection rates for the chosen tracers; first when both dyes are injected into the same site, then when they are injected separately in different groups of animals. If there are N neurones projecting to a site which has been injected with a mixture of two tracers, ([N b - N m ] labelled with true blue alone, Ny labelled with diamidino yellow alone and No labelled with both, i.e. double labelled), then the detection rates for true blue (Rb) and diamidino yellow (Ry) are given by: R b = ( [ N b - Nm] + N d ) / N
(V)
Ry = (Ny + N d ) / N
(8)
The detection rate for doubly labelled neurones will be the conjoint detection rate of both tracers, given by: R b .Ry = Nd/N
(9)
By substitution from Eqn. 9, the detection rates become: Rb = N d / ( Ny + gd)
(10)
R y = N d / ( N b - N m + Nd)
(11)
Detection rates and branched neurones
Once the detection rates have been determined from injections of mixtures into the same site, they can be used to correct the number of branched neurones counted after single injections into two sites. After the number of such profiles has been
180 corrected for both size and misidentification of doubly labelled cells (as described previously), the number of cell centres (C b for true blue, Cv for diamidino yellow and Cd for doubly labelled neurones) is corrected for detection rates as follows. Ch = (Nh -- N m ) / R h
(12)
(\. = N,,/R,
(13)
Cd = Nd/( R h • Ry )
(14)
A short computer program incorporating the corrections is available from the authors.
Experimental determination of detection rates Data obtained from labelling cells of the olivocerebellar pathway with a mixture of tracers injected at one site will now be presented. The various corrections will be applied in sequence in Table II. Correction for size. The rostrocaudal lengths of nuclei and cytoplasm for singly and doubly labelled cells were determined and found to be the same in neurones of this particular pathway (Table I). The relative lengths of nuclei and cytoplasm obtained indicate that cytoplasmic tracers will label 16% more profiles (in the 30/~m thick sections used) than nuclear tracers. The number of profiles labelled with each tracer was counted (column 1, Table II). The mean number of cell centres in a section (column 2) was derived from the number of profiles courted by applying Eqn. 2 to true blue labelled neurones and Eqn. 3 to diamidino yellow labelled and doubly labelled neurones. Correction for misidentified doubly labelled cells. After injecting a mixture of dyes, the mean number of nuclear profiles observed was 207 (Table II). The number of doubly labelled cell centres, obtained from applying Abercrombie's correction to the number of doubly labelled nuclear profiles, was 145 (Eqn. 4). If the cytoplasmic profiles of these 145 cell centres could also have been correctly identified, then inverting Abercrombie's correction gives the number of expected cytoplasmic profiles as 241 (Eqn. 5). Consequently, 34 (241-207) cytoplasmic profiles were labelled blue, but their nuclei were out of the plane of the section and were labelled with diamidino yellow. This corresponds to 20 missed cell centres (Eqn. 6) which belonged to doubly labelled cells but were apparently labelled only by true blue. It follows that 19 ( 3 9 - 20) centres belonged to cells singly labelled with true blue (column 3, Table II).
TABLE 1 THE MEAN (4- S.E.M.) ROSTROCAUDALLENGTH IN /~m OF FLUORESCENT LABELLED NEURONES IN THE MEDIAL ACCESSORYOLIVE OF ONE RAT (n = 14) Singly labelled Doubly labelled
Nuclei
Cytoplasm
13.1 _+0.2 12.84-0.5
20.34-0.4 20.3 + 0.7
181 TABLE II NUMBERS OF LABELLED PROFILES AND CELL CENTRES The numbers are the totals (+_ S.E.M.) obtained from both sides of every sixth section through the medial accessory olive, averaged across animals. Number of animals Mixture injection TB DY DL Single injection TB DY
Profiles
Cell centres (corrected for size)
Cell centres (corrected for misssed DL)
65.3+ 9.0 2.7_+ 0.6 207.2 _+22.7
39.2_+ 5.4 1.9_+ 0.4 1'44.5+ 15.9
19.0_+ 3.7 1.9_+ 0.4 144.5 _+15.9
304.2 _+53.3 180.0 _+32.9
182.9 _+31.8 125.7 _+23.0
6
8 7
D e t e c t i o n rates. Eqns. 10 and 11 were applied to the data in Table II. The detection rate for true blue (R b) was 0.986 + 0.003, and for diamidino yellow ( R y ) it was 0.886 + 0.014. I n t e r a c t i o n b e t w e e n tracers
The most frequent observation in the literature is that the presence of one tracer m a y mask another (Ad6r et al., 1980; De Olmos and Heimer, 1980; Swanson, 1983; Alheid et al.,, 1984). In the present experiments, after injections of a mixture of two tracers, the total n u m b e r of profiles labelled with true blue was 272 (207 + 65), and with diamidino yellow, 210 (207 + 3). For comparison, tracers were injected singly in the same site in different animals. After these single tracer injections (Table II), the mean n u m b e r of profiles labelled with true blue was 304, and with diamidino yellow, 180. Thus more profiles were labelled with true blue and fewer with diamidino yellow. This raises the possibility that diamidino yellow in the nuclei of double labelled cells might have absorbed light emitted by true blue in the surrounding cytoplasm, i.e. that scintillation was occurring (Bj0rklund and Skagerberg, 1979). True blue emits light of a wavelength capable of exciting diamidino yellow, so that true blue in the cytoplasm may increase the excitation of diamidino yellow in the nucleus, thereby increasing the detection rates. This hypothesis was tested by exciting singly and doubly labelled cells, first with ultra-violet light of 340-380 nm (which excites both true blue and diamidino yellow), then with short wavelength visible light of 400-425 nm (which excites mainly diamidino yellow). A second group of cells was excited first with visible light then with ultra-violet light, to control for order effects. The background or cytoplasmic levels were subtracted from each reading then the ratio of the intensity emitted with ultra-violet excitation to that with visible light excitation was calculated. N o significant differences were found in relation to the order in which visible and ultra-violet light were used, so the two groups (visible light first and ultra-violet light first) were combined.
182
2.0
1.6
~
:6 ~ 1.2
-
~ 0.8 n 0,4
0.0
.
.
.
DL
. DY
. DY
DL
(a)
(b)
Fig. 2. Photometric m e a s u r e m e n t s o b t a i n e d from the nuclei of d o u b l y labelled ( D E ) and singly labelled ( D Y ) neurones, a: c y t o p l a s m i c m e a s u r e m e n t subtracted from nuclear m e a s u r e m e n t , b: b a c k g r o u n d m e a s u r e m e n t s u b t r a c t e d from nuclear measurement. O p e n bars: ultra-violet excitation. Filled bars: short w a v e l e n g t h visible excitation. A r b i t r a r y units.
2.4--
e.D
•o ¢1
2.0-
o~
1.6
¢' i,.
m
•
•
• $
"~
L+
& 4',,
1.2-
"," II
0 ,+,., "~
0
"=
--
0.8
0.4
a:
0.0
%
1
o
DL
DY (a)
DL
DY (b)
Fig. 3. Ratios of photometric measurements obtained from nuclei with ultra violet and visible light excitation, a: cytoplasmic measurement subtracted from nuclear measurement, b: background measurement subtracted from nuclear measurement. DL, doubly labelled neurones; DY. singly labelled nuclei.
183 In doubly labelled neurones the quantity of light emitted from nuclei relative to cytoplasm with ultra-violet excitation (Fig. 2) was greater than with short wavelength visible excitation. For diamidino yellow nuclei the opposite was true. The median ratio of emitted light with ultra-violet excitation to emitted light with short wavelength visible excitation was 75% greater for doubly labelled than for diamidino yellow neurones (Fig. 3). The ratios were subjected to a M a n n - W h i t n e y U-test, which showed a highly significant difference between the doubly labelled and diamidino yellow neurones ( U = 5, P < 0.001). When the light emitted from the nuclei was measured relative to background levels, a similar difference was found (U = 3, P < 0.001). Thus more light was emitted from diamidino yellow in the nuclei of neurones which contained true blue in the surrounding cytoplasm when they were excited by ultra-violet light than when short wavelength visible light was used. This was despite the use of a filter to block the light emitted by true blue itself, and was still true even if the emission from the cytoplasm was first subtracted. This may explain why, when the number of true blue labelled profiles decreased after injections of a mixture of two tracers, the number of diamidino yellow profiles increased. Fortunately the difference in the number of profiles counted after mixture and single tracer injections in our experiments did not reach statistically significant levels (X 2 = 3.76, df = 1), suggesting that the detection rates were not substantially altered by the presence of a second tracer. Thus, although interactions of fluorescent intensity were occurring, the initial levels were sufficiently above visual threshold for this interaction not to affect the counts significantly. Two sets of detection rates must be used if significant interactions are found, one set applying to doubly labelled neurones where interactions occur, and the second set to singly labelled neurones.
Experimental determination of proportion of branched neurones in the olivocerebellar pathway As an illustration of the present method, 2% true blue was injected into the posterior lobe of the cerebellar vermis and 2% diamidino yellow was injected into the anterior lobe. The posterior injections were in lobules VI and VII and the anterior injections were in lobules IV and V (Fig. 4). Only injections which had their maximum extent in the same sagittal plane without overlap of the two tracers were accepted (Payne et al., 1985). Data obtained from the medial accessory olive gave the numbers of branched and unbranched neurones presented in Table III. Three corrections were used. The first, from row 1 to row 2, corrects for the difference in size of nuclei and cytoplasm and reduces the difference between the percentages of true blue and diamidino yellow labelled neurones. The second, from row 2 to row 3, corrects for centres belonging to doubly labelled cells being counted as true blue labelled cytoplasmic profiles. The third, rows 3-4, corrects for the different detection rates of the two tracers. Since these injections were in a similar site to that used earlier in mixture injections, the same detection rates were assumed to apply. The proportion of branched neurones, underestimated by raw profile counts, was increased by 18.5%. Of this total increase,
184 Diamidlno Yellow
ANTERIOR
POSTERIOR
Fig. 4. A section through the cerebellar vermis demonstrating the extent of the injection sites in an animal used to determine numbers of branched neurones.
48.4% was d u e to the c o r r e c t i o n for size. A f u r t h e r 9.2% o f the i n c r e a s e was d u e to the c o r r e c t i o n for d o u b l y l a b e l l e d n e u r o n e s m i s i d e n t i f i e d as true b l u e l a b e l l e d cells b e c a u s e their nuclei w e r e n o t in the p l a n e of the section, a n d 42.4% was d u e to the d i f f e r e n c e in d e t e c t i o n rates of true b l u e a n d d i a m i d i n o yellow. Variability of numbers detected. It is e v i d e n t f r o m the tables that there is c o n s i d e r a b l e v a r i a t i o n in the n u m b e r of p r o f i l e s l a b e l l e d w i t h a p a r t i c u l a r tracer. A f t e r i n j e c t i o n s o f m i x t u r e s , the c o e f f i c i e n t of v a r i a t i o n in the n u m b e r of p r o f i l e s was 27.7% for all true b l u e cells a n d 26.3% for all d i a m i d i n o y e l l o w cells, w h e r e a s the
TABLE III MEAN NUMBERS (AND S.E.M.) OF BRANCHED NEURONES PROJECTING TO TWO SITES IN THE CEREBELLAR VERMIS, SUMMED OVER 6 SECTIONS THEN AVERAGED ACROSS 5 ANIMALS (n = 5) True blue Profiles Cell centres Correction for: Size Missed DL Detection rate
Doubly labelled
Diamidino yellow
No.
%
No.
No.
%
164.8 (49.0)
58.8
27.8 (7.7)
9.9
8"/.6 (10.9)
31.3
98.9 (29.4) 96.2 (29.1) 97.5 (29.5)
55.1 54.4 51.7
19.4 (5.4) 19.4 (5.4) 22.2 (6.1)
10.8 11.0 11.8
61.1 ("/.6) 61.1 ('/.6) 69.0 (8.6)
34.1 34.6 36.6
%
185 coefficients for the detection rates were 0.8% and 3.7% respectively. Clearly, the detection rate is a robust measure whereas the absolute number of profiles is very variable. The most likely explanation is variation in injection size between animals.
Discussion The use of two retrogradely transported fluorescent tracers was one of the first anatomical techniques to identify branched neurones qualitatively (Van der Kooy et al., 1978). The present paper suggests an analytical procedure to quantify the proportion of branched neurones in a population of cells projecting to two injection sites. The corrections which have to be applied to the number of profiles counted in order to convert them into relative numbers of neurones involve 3 stages. The first two stages depend on knowing the size of branched and unbranched neurones and their nuclei. Clearly, nuclear and cytoplasmic profiles differ in size, and unless corrections for this are employed, errors will be made. It is interesting to note that, in their criticism of the double labelling technique, Alheid et al. (1984b) found no significant difference in the number of cells labelled with nuclear yellow and granular blue after unilateral injections in 26/~m thick sections despite the fact that one tracer labels nuclei while the other labels cytoplasm. This suggests that there were differences in the detection rates of the tracers they used, or that leakage had occurred. Branched neurones may differ in size from unbranched neurones so there will be an error of estimation if the two are not distinguished. This is an unavoidable error if the single tracer algebraic technique of Alheid et al. (1984a, 1984b) is used. It is therefore essential that branched neurones are identified individually to assess their relative size, and this requires double labelling. In the method presented here, stage I is a conventional correction for different sizes of the cellular compartments labelled by two tracers. In the present experiments, this correction accounted for 48.4% of the total increase in the relative number of branched neurones. Stage II reclassifies a proportion of cytoplasmically labelled profiles as being double labelled but with their nuclei out of the plane of the section. The final correction, stage III, takes into account the different detection rates of the two tracers. This accounted for 42.4% of the total increase. It is sometimes assumed that true blue is detected more frequently than diamidino yellow because it diffuses further at the injection site. After the two stereological corrections of stage I and II, the detection rates of the two tracers were within 10% of each other (99% and 89% respectively). Other factors contributing to differences in detection rate, besides diffusion, are: the placement, volume and concentration of tracers; the affinity for uptake of the tracers; the efficiency and rate of retrograde transport; the fluorescent intensity of the tracers; leaking from the cells; masking and scintillation. One effect of injection site placement was obviated by the selection of only those cases in which the maximum extent of the injections was in the same sagittal plane
186 and there was no overlap between them. The possible effect of different sites on detection rates, such as unequal distances to travel, was avoided by determining the rates after injections in the same place as in the experimental groups. Of the other factors mentioned, only masking and scintillation were specifically investigated in the present experiments. The nuclei of singly labelled diamidino yellow neurones emitted more light when short wavelength visible excitation was used than when they were excited by ultra-violet light, as predicted from the absorption spectrum of diamidino yellow. However, photometric measurements indicated that a diamidino yellow labelled nucleus emitted more light with ultra-violet excitation if there was true blue in the surrounding cytoplasm. This strongly suggests that true blue emissions were secondarily exciting diamidino yellow. The emission spectrum of true blue is coextensive with the absorption spectrum of diamidino yellow, so that if cells were doubly labelled their nuclei fluoresced more intensely than if they were singly labelled. However, although the number of profiles labelled by diamidino yellow was 16.6% greater after injections of mixtures than after single tracer injections, the increase was not substantial enough to affect the detection rate of this tracer significantly. This implies that the intensity of fluorescence of singly labelled nuclei was already above visual threshold, or that the variation between animals was too great to reveal the effects of scintillation and masking. The tendency of diamidino yellow to mask true blue (by absorbing its emissions), indicated by a 10.4% reduction, was not statistically significant either. A possible explanation of why Alheid et al. observed so much masking is that there was extensive leakage from transporting cells to their neighbours, giving them spuriously high counts. If, however, there is a significant interaction between tracers, it is possible to allow for this by incorporating the altered detection rates in the equations presented here. The absolute number of neurones labelled by retrograde tracers is notoriously variable. In the present experiments, coefficients of variation of 25% or more were obtained, in line with the results of other investigators (Alheid et al., 1984b; Cavada et al., 1984; Huisman et al., 1983; IUert et al., 1982; Luskin and Price, 1982; Room et al., 1981; Sawchenko and Swanson, 1981). Given this variation, any technique which relies on absolute counts requires large numbers of animals to obtain reliable results. This drawback applies to the algebraic method of Alheid et al. (1984a), where, to detect the small proportion of branched neurones obtained here, one would require approximately 20 animals for each comparison. By contrast, the coefficients of variation for the detection rates of the two tracers were less than 4% across animals. It is inherently more satisfactory to use each animal as its own control whenever possible and the double labelling technique permits exactly this. At present, therefore, quantification gives the relative, but not the absolute, numbers of branched neurones in a population. In addition to the practical problems involved in implementing the algebraic approach (Alheid et al., 1984a, 1984b) discussed above, there is a theoretical limitation to their technique, making it applicable only if the detection rate is 100%. The derivation given by Alheid et al. for a detection rate of 80% contains an error which cannot be avoided unless the detection rates are known. The error in their derivation is as follows: K a is the number of profiles labelled by an injection of a
187 single tracer into one of two sites, K b the number after injection into the other site and Kab is the number after injection of the same tracer into both. The number of cells projecting to the two sites is A and B and to both sites, AB. The equations they gave were: K a = 80%A + 80%AB
(a)
80%AB + 80%B
(b)
Kb=
Kab = 8 0 % A + 8 0 % A B + 8 0 % B
(c)
From (a-c) they derived the proportion ( P ) of branched neurones: P = AB/(A
+ AB + B)
(d)
The errors occurs in Eqn. c. Of the branched neurones, 80% would be labelled from site A, but a further 80% of the neurones unlabelled from site A (20%) would be labelled from site B (i.e. 16%), making 96% (80% + 16%) altogether. Thus Eqn. c should be: Kah = 8 0 % A + 9 6 % A B + 8 0 % B
(e)
Using this corrected equation, the number of branched neurones is given by Ka + K b - Kab, o r ( 8 0 + 80 -- 96)%AB. Thus the proportion of branched neurones becomes: P = 6 4 % A B / ( 8 0 % A + 96%AB + 80%B)
(f)
It can be seen that the proportion of branched neurones cannot be calculated until the detection rate is known. This is a serious constraint for the algebraic approach because, of source, there is no way of knowing the detection rate for a single tracer technique. It is only when double labelling is employed that detection rates (including the effect of interaction on these) can be measured. In applying a quantitative method, it is important not to use it outside its limitations, and to bear in mind the difference between relative and absolute numbers. This paper provides a quantitative analysis of the proportion of branched neurones, correcting for stereological errors and differences in detection rate of the fluorescent tracers. It is perhaps reassuring that after the corrections employed, the final proportion of branched neurones increased by a relatively small amount. For neurones of different size, counted in sections of different thickness, the changes resulting from the present corrections may be much larger. If, therefore, it is of importance to have accurate results, then the present corrections are recommended. On the other hand, the present results indicate that raw profile counts may be sufficient for many purposes. Finally, the location of branching neurones can be determined only by using a double labelling technique. In some cases branched neurones occupy a distinct location (Payne et al., 1985). By providing quantitative information on both the location and the proportion of neurones which branch, the double retrograde tracer technique contributes greatly to the unravelling of neuronal circuitry.
188
Acknowledgements The authors would like to thank C.J. Hill and K. Crawford for their technical assistance. We would also like to thank S.M. Wharton for contributing to the counts in Table III.
References Abercrombie, M. (1946) Estimation of nuclear populations from microtome sections, Anat. Rec.. 94: 239-247. Ad6r, J-P., Room, P., Postema, F. and Korf, J. (1980) Bilaterally diverging axon collaterals and contralateral projections from rat locus coeruleus neurons, demonstrated by fluorescent retrograde double labeling and norepinephrine metabolism, J. Neural Transm., 49: 207-218. Alheid, G.F., Carlsen, J. and Heimer, L. (1984a) An algebraic approach to the detection of multiple markers in complex neuronal systems, J. Neurosci. Meth., 10: 71-77. Alheid, G.F., Carlsen, J., de OImos, J. and Heimer, L. (1984b) Quantitative determination of collateral anterior olfactory nucleus projections using a fluorescent tracer with an algebraic solution to the problem of double retrograde labeling, Brain Res.. 292: 17-22. Aschoff, A. and Hollander, H. (1982) Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). I. A parametric study in the central visual system of the adult rat, J. Neurosci. Meth., 6: 179-197. Bentivoglio, M., Kuypers, H.G.J.M. and Catsman-Berrevoets, C.E. (1980) Retrograde neuronal labeling by means of bisbenzimide and nuclear yellow (Hoechst 769121). Measures to prevent diffusion of the tracers out of retrogradely labeled neurons, Neurosci. Lett., 18: 19-24. Bjorklund, A. and Skagerberg, G. (1979) Simultaneous use of retrograde fluorescent tracers and fluorescence histochemistry for convenient and precise mapping of monoaminergic projections and collateral arrangements in the C.N.S., J. Neurosci. Meth., 1: 261-277. Carpenter, M.B. (1984) Interconnections between the corpus striatum and brain stem nuclei. In J.S. McKenzie, R.E. Kemm and L.N. Wilcock (Eds.), The Basal Ganglia, Structure and Function, Plenum Press, New York. Cavada, C., Huisman, A.M. and Kuypers, H.G.J.M. (1984) Retrograde double labeling of neurons: the combined use of horseradish peroxidase and diamidino yellow dihydrochloride (DY. 2HC1) compared with true blue and D1Y.2HCI in rat descending pathways, Brain Res., 308: 123-136. DeHoff, R.T. and Rhines, F.N. (1961) Determination of the number of particles per unit volume from measurements made on random plane sections: the general cylinder and ellipsoid, Trans. Am. Inst. Mineral Met. Eng., 221: 975-982. De Olmos, J. and Heimer, L. (1980) Double and triple labeling of neurons with fluorescent substances: the study of collateral pathways in the ascending raphe system, Neurosci. Left., 19: 7-12. Henderson, Z. (1981) Ultrastructure and acetylcholinesterase content of neurones forming connections between the striatum and substantia nigra of rat, J. Comp. Neurol., 197: 185-196. Huisman, A.M., Kuypers, H.G.J.M., Conde, F. and Keizer, K. (1983) Collaterals of rubrospinal neurons to the cerebellum in the rat. A retrograde fluorescent double labeling study, Brain Res., 264: 181-196. Illert. M., Fritz, N., Aschoff, A. and Hollander, H. (1982) Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). II. A parametric study in the peripheral motor system of the cat, J. Neurosci. Meth., 6: 199-218. Keizer, K., Kuypers, H.G.J.M., Huisman, A.M and Dann, O. (1983) Diamidino yellow dihydrochloride (DY. 2HCI); a new fluorescent retrograde neuronal tracer which migrates only very slowly out of the cell, Exp. Brain Res., 51: 179-191. Kristensson, K., Olsson, Y. and Sjostrand, J. (1971) Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain R.es., 32: 399-406. Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Padt, R.E. (1977) Retrograde axonal transport of fluorescent substances in the rat's forebrain, Neurosci. Lett., 6: 127-135.
189 Kuypers, H.G.J.M., Bentivoglio, M., Van der Kooy, D. and Catsman-Berrevoets, C.E. (1979) Retrograde transport of bisbenzimide and propidium iodide through axons to their parent cell bodies, Neurosci. Lett., 12: 1-7. Kuypers, H.G.J.M., Bentivoglio, M., Catsman-Berrevoets, C.E. and Bharos, A.T. (1980) 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: 383-392. Lenn, N.J., Wong, V. and Hamill, G.S. (1983) Left-right pairing at the crest synapses of rat interpeduncular nucleus, Neuroscience, 9: 383-389. Luskin, M.B. and Price, J.L. (1982) The distribution of axon collaterals from the olfactory bulb and the nucleus of the horizontal limb of the diagonal band to the olfactory cortex, demonstrated by double retrograde labeling techniques, J. Comp. Neurol., 209: 249-263. Payne, J.N., Wharton, S.M. and Lawes, I.N.C. (1985) Quantitative analysis of the topographical organization of the olivo-cerebellar projections in the rat, Neuroscience, 15: 403-415. Preston, R.J., Bishop, G.A. and Kitai, S.T. (1980) Medium spiny neuron projection from the rat striatum: an intracellular horseradish peroxidase study, Brain Res., 183: 253-263. Room, P., Postema, F. and Korf, J. (1981) Divergent axon collaterals of rat locus coeruleus neurons: demonstration by a fluorescent double labeling technique, Brain Res., 221: 219-230. Sawchenko, P.E. and Swanson, L.W. (1981) A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemical techniques, Brain Res., 210: 31-51. Swanson, L.W. (1983) The use of retrogradely transported fluorescent markers in neuroanatomy. In J.L. Barker and J.F. McKelvy (Eds.), Current Methods in Cellular Neurobiology. Volume 1. Anatomical Techniques, Wiley, New York, pp. 219-240. Van der Kooy, D., Kuypers, H.G.J.M. and Catsman-Berrevoets, C.E. (1978) Single mamillary body cells with divergent axon collaterals. Demonstration by a simple, fluorescent retrograde double labeling technique in the rat, Brain Res., 158: 189-196.