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Differences in fluorogold and wheat germ agglutinin-horseradish peroxidase labelling of bladder afferent neurons
352
Brain Research, 613 (1993) 352-356 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25686
Differences in Fluorogold and wheat germ agglutinin-horseradish peroxidase labelling of bladder afferent neurons M.N. Kruse, S.L. Erdman, G. Puri and W.C. de Groat Department of Pharmacology, Unicersity of Pittsburgh, Pittsburgh, PA 15261 (USA) (Accepted 9 March 1993)
Key words: Neuronal tracer; Histology; Tissue dehydration
Rat urinary bladder afferent neurons were significantly smaller (34%) when labelled with Fluorogold (FG) than when labelled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP). This study showed that this difference was due to an artifact of tissue processing (ethanol dehydration) and was not due to uptake and transport of the two tracers by two different subpopulations of bladder afferents.
Axonal transport of tracer substances has been used to identify the afferent and efferent neurons innervating various organs. In the course of examining retrogradely labelled bladder afferent cells in the dorsal root ganglia (DRG), it was noted that the average cross-sectional area of the cell profiles differed depending on whether Fluorogold (FG) or wheat germ agglutinin-horseradish peroxidase ( W G A - H R P ) was used. Since these tracers have recently been used to study the changes in visceral afferent neuron size in lumbosacral dorsal root ganglia in response to various pathological conditions 5'7"8, it is important to know the reasons for the measured differences in profile areas with the two tracers. The purpose of this study was to determine whether the noted difference reflects: (1) a true difference in size due to different populations of neurons with different mean sizes preferentially taking up one or the other tracer or (2) an artifact of the handling or processing of the tissue. If FG and W G A HRP labelled two different subpopulations of bladder afferents, this would have important ramifications for labelling studies using either tracer. Alternatively, if the observed difference was an artifact, this would indicate that the measured areas might not be accurate, and it would be important to determine whether they could be corrected. These questions were addressed here by (1) utilizing FG and W G A - H R P dou-
ble-labelling studies of the bladder to determine if individual bladder afferents can transport both tracers and (2) examining the effect of various-tissue processing methods on the measured cross-sectional area of bladder D R G cells. Female Wistar rats (245-300 g) were anesthetized with halothane and their urinary bladders injected with FG (4% w / v in distilled H 2 0 , Fluorochrome, Inc.) a n d / o r W G A - H R P (0.5% w / v in distilled H 2 0 , Sigma). Injections totalling 40 /xl were made at 8-10 sites in the dorsal, rostral bladder wall with a 100-1xl Hamilton syringe and a 30-g needle. Following the injection, the needle was kept in place for 30 s, then the site was swabbed with a cotton applicator and rinsed with saline. Four days after W G A - H R P injection and 10-11 days after FG injection, the animals were deeply anesthetized with pentobarbital ( ~ 40 m g / k g i.p.), and perfused with Ringers solution followed by 4% paraformaldehyde fixative (FG injections) or 1% paraformaldehyde and 1% glutaraldehyde fixative ( W G A - H R P and combined F G / W G A - H R P injections). The L 6 and S~ DRGs, which contain cells providing a major part of the bladder afferent innervation ~'9, were post-fixed, rinsed in phosphate buffer, and placed in 10%, 20% and 30% sucrose prior to freezing and cryostat sectioning at 28-1zm thickness. The sections
Correspondence: M.N. Kruse, Department of Pharmacology, E1303 BST, University of Pittsburgh, Pittsburgh, PA 15261, USA.
353 were directly mounted on gelatinized slides and processed by one of the following three methods: the H R P protocol, the F G protocol, or D M S O / g l y c e r i n mounting. In the F G protocol, the slides with freshly mounted tissue were heat dried (43°C) for 5-10 min, and put through the following dehydration and de-fatting sequence: distilled H 2 0 (5 min), ethanol: 35%, 70%, 2 × 95%, 2 X 100% baths (2-3 min each) and 3 xylene baths (2-3 rain each). The slides were coverslipped with Entellan. In the H R P protocol, the slides with freshly mounted tissue were heat dried (43°C) for 5-10 min, and processed by the tetramethylbenzidine method 6, after which the slides were put in 100% ethanol for 1-2 min and xylene for 2-3 min and coverslipped with Permount. For DMSO or glycerin mounting, the slides were heat dried and coverslipped with either DMSO or glycerin. The cross-sectional area of all labelled cell profiles were measured in every third tissue section (to eliminate double measurement of the same cell) with the Olympus Cue-2 image analysis system and a mean profile cross-sectional area was calculated for each rat. The mean profile area from all the FG-injected rats or the W G A - H R P - i n j e c t e d rats was used to calculate an overall mean F G profile area or W G A - H R P profile area. The number of dye-labelled cells was not calculated due to a large variability obtained in the number of labelled profiles between rats. This variability may be attributable to the imprecision inherent in manual injection of dye into 8-10 locations in the bladder muscle. To evaluate co-labelling of bladder D R G cells, the right side of the bladders of 3 rats were injected with 40/zl of both FG and W G A - H R P 11 days (FG) and 4
days ( W G A - H R P ) prior to perfusion. The bladder afferent neurons in the right L 6 and S 1 DRGs from these rats were then examined for double labelling by examining HRP-processed sections under both darkfield and ultraviolet (340-380 nm wavelength) illumination. Processing-induced tissue shrinkage was assessed as follows. Shrinkage induced by the FG protocol was measured in 2 rats by mounting some freshly cut D R G sections with DMSO or glycerin, coverslipping, and measuring the cross-sectional areas of individually identified profiles (pre-dehydration measurements). The coverslips were then removed by soaking in 0.1 M phosphate buffer, and the slides were put through the normal dehydration/defatting sequence of the FG protocol. The area of the previously identified profiles were remeasured (post-dehydration measurements). In addition, these data were used to test the correlation between the initial profile area and the amount of dehydration-induced shrinkage. FG-labelled D R G cells were also examined for evidence of tissue shrinkage by processing alternating sections via either the H R P or FG process, then measuring the areas of FG-labelled profiles following each type of processing. The t-test was used for statistical analysis. Results are given as mean :]: S.E.M. and differences between means are indicated as statistically significant if P < 0.05. The cross-sectional area of bladder D R G cell profiles labelled with FG were significantly smaller (34% decrease) than those labelled with W G A - H R P (387 _+ 29 tzm 2, n = 7 animals vs. 583 _+ 22 /zm 2, n = 3 animals) (Fig. 1). Colocalization of F G and W G A - H R P label was
Size of bladder DRG cells 12
i°8
0
.0
• FG ~ WGA-HRP
!
................. 2{D 400 61}0
800
1{}00
L 12{D
.., 1400
.. 16(10
area of DRG cell profiles (urn2) Fig. 1. Frequency distribution of FG and WGA-HRP-labelled bladder afferent neurons. The range of measured areas of FG-labelled D R G cell profiles (dehydrated) was smaller than that of WGA-HRP-labelled D R G profiles.
354 600 ,5oo :zk
t~
100 0
o Fig. 3. Effect of dehydration on cell profile cross-sectional area. There was no significant difference in the area of HRP and non-dehydrated FG profiles ( F G / n o dehydr). Following dehydration of the FG cells via the FG protocol ( F G / F G dehydr), there was a significant decrease in profile area, and a further decrease in area occurred in FG profiles dehydrated via the HRP protocol ( F G / H R P dehydr). i
Fig. 2. W G A - H R P (top) and FG (bottom) are co-localized in bladder afferent neurons (arrowheads). W G A - H R P appears to quench fluorescence in some cells. Arrow indicates a WGA-HRP-labelled cell which appears as a hole in the tissue under UV light. B a r 50/~m.
observed in individual D R G cells in all 3 animals tested with double injections (Fig. 2, arrowheads). However, fewer than half of all labelled D R G cells were co-labelled. There appeared to be some interference between the two tracers as some cells containing H R P reaction product appeared as holes in the tissue when visualized under fluorescence (Fig. 2, arrow). The FG dehydration process resulted in a 38% decrease in the area of FG-labelled cell profiles. Prior to dehydration when mounted in glycerin or DMSO, 34 individually identified F G profiles measured 533 _+ 36 p~m2, whereas after dehydration via the FG protocol the same profiles measured 332_+ 24 ~zm2 (Fig. 3). There was no correlation between the initial profile area and the amount of shrinkage of each profile (Fig. 4). The cross-sectional areas of W G A - H R P cell profiles (processed via the H R P protocol) were not significantly different from the area of F G - l a b e l l e d / n o n - d e hydrated profiles. However, the average area of FGfilled profiles which underwent FG dehydration was significantly smaller than the FG labelled/non-dehydrated profiles. The FG cell profiles which underwent H R P dehydration (without undergoing the tetramethylbenzidine reaction process) were significantly smaller (300 _+ 2 6 / ~ m 2) than the other groups (Fig. 3). This study demonstrated that the difference in cross
sectional areas of bladder afferent neuronal profiles labelled with W G A - H R P and FG is due to post-fixation processing and dehydration and not to differential transport of the tracers in different populations of neurons. It was shown here that a given bladder sensory neuron can carry both FG and W G A - H R P , and that the observed 34% reduction in FG-labelled profile area as compared to W G A - H R P - l a b e l l e d profile area can be accounted for by the FG dehydration sequence. These results caution that when size comparisons are made between cells filled with different tracers, a careful analysis of the tissue processing methods must be
100
=
80O m
m [] []
[]
[] E m
n
D
tlm
[]
m
0
D
t~
m
tl la o
D
[]
m
[]
[]
m
~a
~0
1000
1200
Initial profile area Fig. 4. Amount of dehydration-induced shrinkage is unrelated to initial profile area. There was a wide variation in the amount of dehydration-induced shrinkage in FG-labelled profiles which was not correlated with the initial (pre-dehydration) profile area.
355 done to ensure that observed differences are not artifactual. The FG/WGA-HRP double-labelling studies showed that at least some of the bladder sensory neurons can carry both tracers. The failure of all of the neurons to exhibit double labelling is possibly due to a subpopulation of bladder neurons which only transport one tracer. However, we think it is more likely that the non-double-labelled neurons are capable of transporting both tracers, but do not show double labelling because of (1) non-overlapping or only partially overlapping injections of the two tracers, resulting in some singly labelled cells a n d / o r (2) quenching of F G fluorescence by W G A - H R P in some double-labelled cells 2 (see also Fig. 2). Thus, the actual number of doublelabelled neurons in this study may have been greater than what was observed, as heavily filled W G A - H R P cells appeared to interfere with the visualization of FG-labelled cells using fluorescence microscopy. An additional argument that all populations of bladder afferents (i.e. A~ and C-fibers) 9 can transport both F G and W G A - H R P is that the mean profile area of W G A - H R P cells (583 + 22 /zm 2) and non-dehydrated FG cells (533 + 36/xm 2) falls between the mean profile area of A~ (702 + 158 /zm 2) and C-fibers (449 + 47 /xm2) 4, which would indicate transport by both fiber groups. It may be a general rule that all peripheral afferents can transport both tracers as a recent study showed that both FG and W G A - H R P label all the sciatic nerve afferents in the rat, and therefore both are transported by all of the afferent subpopulations ( A a through C fibers) in the sciatic nerve 2. It should be noted that this other study applied the tracers to the cut sciatic nerve, therefore demonstrating transport by all fiber types, but not necessarily uptake by terminal endings. The FG-labelled cell profiles of the sciatic nerve were significantly smaller (26%) than the W G A H R P cells, which was attributed to dehydration-induced shrinkage 2. Other studies have shown that tissue dehydration does not necessarily give a fixed amount of shrinkage. It was shown that the amount of dehydration-induced brain shrinkage varied by brain region (44-59% shrinkage) 3 and thus a single correction factor for dehydration-induced shrinkage of all tissue types does not exist. It was also reported that ethanol dehydration in brain slices resulted in a shrinkage of Lucifer yellowfilled neurons to less than 2 / 3 of their original size, however, similar to the results obtained here, HRPfilled neurons did not exhibit shrinkage 3. The lack of shrinkage of HRP-filled cells is attributed to the formation of an insoluble precipitate during the H R P
reaction process which does not allow the cell to shrink when exposed to the H R P dehydration process 3. In contrast, it was shown here that FG-filled cells put through the H R P dehydration process actually shrank more than when put through the F G dehydration. This is presumably because the F G protocol used in our experiments had a more gradual change in ethanol concentration than the H R P protocol, resulting in smaller osmotic gradients and thus less tissue shrinkage 3. These results indicate that the method of tissue processing can lead to large artifactual changes in cell profile area. Thus a comparison of cell measurements from different studies must take into account methodology (e.g. ethanol dehydration sequence). Several recent studies have examined changes in cross-sectional area of specific neuronal populations due to various experimental perturbation 5'7'8. For example, urethral obstruction-induced hypertrophy of bladder post-ganglionic and D R G cells8; whereas spinal cord injury induced hypertrophy only of bladder D R G cells 5. It was demonstrated here that the initial profile area was unrelated to the degree of shrinkage of an individual cell indicating that large cells were not more apt to shrink than small ones. This indicates that the % change in neuronal cross-sectional area following a given experimental perturbation should be accurate even in dehydrated tissue. While it might be argued that DMSO or glycerinmounting of FG-labelled sections should routinely be done to avoid dehydration artifact, DMSO and glycerin mounting are less satisfactory than dehydration/Entellan mounting for long-term storage. However, if the absolute area of fluorescent dye-labelled cells is important, DMSO or glycerin mounting could be used to calculate a correction factor for that tissue, and if necessary, the coverslips then removed and the slides put through the normal FG dehydration process. In summary, all urinary bladder afferents appear to take up and transport both FG and W G A - H R P . However, the measured cross-sectional area of FG-labelled cell profiles was 34% less than that of W G A - H R P labelled profiles due to ethanol dehydration. This project was supported by a grant from the PVA Spinal Cord Research Foundation to M.N.K. and NIH Grant DK372421 to W.C.d.G. 1 Applebaum, A.E., Vance, W.H. and Coggeshall, R.E., Segmental localization of sensory cells that innervate the bladder, J. Comp. Neurol., 192 (1980) 203-209. 2 Baranowski, A.P., Anand, U. and McMahon, S.B., Retrograde labelling of dorsal root ganglion cells in the rat: a quantitative and morphological comparison of Fluoro-Gold with horseradish peroxidase labelling, Neurosci. Lett., 141 (1992) 53-56.
356 3 Grace, A.A. and Llinas, R., Morphological artifacts induced in intracellularly stained neurons by dehydration: circumvention using rapid dimethyl sulfoxide clearing, Neuroscience, 16 (1985) 461-475. 4 Harper, A.A. and Lawson, S.N., Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurones, Z Physiol., 359 (1985) 31-46. 5 Kruse, M.N., Erdman, S.L., Tanowitz, M. and de Groat, W.C., Differential effects of spinal cord injury on the morphology of bladder afferent and efferent neurons, Soc. Neurosci. Abstr., 18 (1992) 127. 6 Mesulam, W.M., The blue reaction product in horseradish peroxidase neurochemistry: incubation parameters and visibility, Z Histochem. Cytochem., 24 (1976) 1273-1280.
7 Steers, W.D., Ciambotti, J., Erdman, S. and de Groat, W.C., Morphological plasticity in efferent pathways to the urinary bladder of the rat following urethral obstruction, Z Neurosci., 10 (1990) 1943-1951. 8 Steers, W.D., Ciambotti, J., Etzel, B., Erdman, S. and de Groat, W.C., Alterations in afferent pathways from the urinary bladder of the rat in response to partial urethral obstruction, J. Comp. Neurol., 310 (1991) 401-410. 9 Vera, P.L. and Nadelhaft, I., Conduction velocity distribution of afferent fibers innervating the rat urinary bladder, Brain Res., 520 (1990) 83-89.
Brain Research, 613 (1993) 352-356 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25686
Differences in Fluorogold and wheat germ agglutinin-horseradish peroxidase labelling of bladder afferent neurons M.N. Kruse, S.L. Erdman, G. Puri and W.C. de Groat Department of Pharmacology, Unicersity of Pittsburgh, Pittsburgh, PA 15261 (USA) (Accepted 9 March 1993)
Key words: Neuronal tracer; Histology; Tissue dehydration
Rat urinary bladder afferent neurons were significantly smaller (34%) when labelled with Fluorogold (FG) than when labelled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP). This study showed that this difference was due to an artifact of tissue processing (ethanol dehydration) and was not due to uptake and transport of the two tracers by two different subpopulations of bladder afferents.
Axonal transport of tracer substances has been used to identify the afferent and efferent neurons innervating various organs. In the course of examining retrogradely labelled bladder afferent cells in the dorsal root ganglia (DRG), it was noted that the average cross-sectional area of the cell profiles differed depending on whether Fluorogold (FG) or wheat germ agglutinin-horseradish peroxidase ( W G A - H R P ) was used. Since these tracers have recently been used to study the changes in visceral afferent neuron size in lumbosacral dorsal root ganglia in response to various pathological conditions 5'7"8, it is important to know the reasons for the measured differences in profile areas with the two tracers. The purpose of this study was to determine whether the noted difference reflects: (1) a true difference in size due to different populations of neurons with different mean sizes preferentially taking up one or the other tracer or (2) an artifact of the handling or processing of the tissue. If FG and W G A HRP labelled two different subpopulations of bladder afferents, this would have important ramifications for labelling studies using either tracer. Alternatively, if the observed difference was an artifact, this would indicate that the measured areas might not be accurate, and it would be important to determine whether they could be corrected. These questions were addressed here by (1) utilizing FG and W G A - H R P dou-
ble-labelling studies of the bladder to determine if individual bladder afferents can transport both tracers and (2) examining the effect of various-tissue processing methods on the measured cross-sectional area of bladder D R G cells. Female Wistar rats (245-300 g) were anesthetized with halothane and their urinary bladders injected with FG (4% w / v in distilled H 2 0 , Fluorochrome, Inc.) a n d / o r W G A - H R P (0.5% w / v in distilled H 2 0 , Sigma). Injections totalling 40 /xl were made at 8-10 sites in the dorsal, rostral bladder wall with a 100-1xl Hamilton syringe and a 30-g needle. Following the injection, the needle was kept in place for 30 s, then the site was swabbed with a cotton applicator and rinsed with saline. Four days after W G A - H R P injection and 10-11 days after FG injection, the animals were deeply anesthetized with pentobarbital ( ~ 40 m g / k g i.p.), and perfused with Ringers solution followed by 4% paraformaldehyde fixative (FG injections) or 1% paraformaldehyde and 1% glutaraldehyde fixative ( W G A - H R P and combined F G / W G A - H R P injections). The L 6 and S~ DRGs, which contain cells providing a major part of the bladder afferent innervation ~'9, were post-fixed, rinsed in phosphate buffer, and placed in 10%, 20% and 30% sucrose prior to freezing and cryostat sectioning at 28-1zm thickness. The sections
Correspondence: M.N. Kruse, Department of Pharmacology, E1303 BST, University of Pittsburgh, Pittsburgh, PA 15261, USA.
353 were directly mounted on gelatinized slides and processed by one of the following three methods: the H R P protocol, the F G protocol, or D M S O / g l y c e r i n mounting. In the F G protocol, the slides with freshly mounted tissue were heat dried (43°C) for 5-10 min, and put through the following dehydration and de-fatting sequence: distilled H 2 0 (5 min), ethanol: 35%, 70%, 2 × 95%, 2 X 100% baths (2-3 min each) and 3 xylene baths (2-3 rain each). The slides were coverslipped with Entellan. In the H R P protocol, the slides with freshly mounted tissue were heat dried (43°C) for 5-10 min, and processed by the tetramethylbenzidine method 6, after which the slides were put in 100% ethanol for 1-2 min and xylene for 2-3 min and coverslipped with Permount. For DMSO or glycerin mounting, the slides were heat dried and coverslipped with either DMSO or glycerin. The cross-sectional area of all labelled cell profiles were measured in every third tissue section (to eliminate double measurement of the same cell) with the Olympus Cue-2 image analysis system and a mean profile cross-sectional area was calculated for each rat. The mean profile area from all the FG-injected rats or the W G A - H R P - i n j e c t e d rats was used to calculate an overall mean F G profile area or W G A - H R P profile area. The number of dye-labelled cells was not calculated due to a large variability obtained in the number of labelled profiles between rats. This variability may be attributable to the imprecision inherent in manual injection of dye into 8-10 locations in the bladder muscle. To evaluate co-labelling of bladder D R G cells, the right side of the bladders of 3 rats were injected with 40/zl of both FG and W G A - H R P 11 days (FG) and 4
days ( W G A - H R P ) prior to perfusion. The bladder afferent neurons in the right L 6 and S 1 DRGs from these rats were then examined for double labelling by examining HRP-processed sections under both darkfield and ultraviolet (340-380 nm wavelength) illumination. Processing-induced tissue shrinkage was assessed as follows. Shrinkage induced by the FG protocol was measured in 2 rats by mounting some freshly cut D R G sections with DMSO or glycerin, coverslipping, and measuring the cross-sectional areas of individually identified profiles (pre-dehydration measurements). The coverslips were then removed by soaking in 0.1 M phosphate buffer, and the slides were put through the normal dehydration/defatting sequence of the FG protocol. The area of the previously identified profiles were remeasured (post-dehydration measurements). In addition, these data were used to test the correlation between the initial profile area and the amount of dehydration-induced shrinkage. FG-labelled D R G cells were also examined for evidence of tissue shrinkage by processing alternating sections via either the H R P or FG process, then measuring the areas of FG-labelled profiles following each type of processing. The t-test was used for statistical analysis. Results are given as mean :]: S.E.M. and differences between means are indicated as statistically significant if P < 0.05. The cross-sectional area of bladder D R G cell profiles labelled with FG were significantly smaller (34% decrease) than those labelled with W G A - H R P (387 _+ 29 tzm 2, n = 7 animals vs. 583 _+ 22 /zm 2, n = 3 animals) (Fig. 1). Colocalization of F G and W G A - H R P label was
Size of bladder DRG cells 12
i°8
0
.0
• FG ~ WGA-HRP
!
................. 2{D 400 61}0
800
1{}00
L 12{D
.., 1400
.. 16(10
area of DRG cell profiles (urn2) Fig. 1. Frequency distribution of FG and WGA-HRP-labelled bladder afferent neurons. The range of measured areas of FG-labelled D R G cell profiles (dehydrated) was smaller than that of WGA-HRP-labelled D R G profiles.
354 600 ,5oo :zk
t~
100 0
o Fig. 3. Effect of dehydration on cell profile cross-sectional area. There was no significant difference in the area of HRP and non-dehydrated FG profiles ( F G / n o dehydr). Following dehydration of the FG cells via the FG protocol ( F G / F G dehydr), there was a significant decrease in profile area, and a further decrease in area occurred in FG profiles dehydrated via the HRP protocol ( F G / H R P dehydr). i
Fig. 2. W G A - H R P (top) and FG (bottom) are co-localized in bladder afferent neurons (arrowheads). W G A - H R P appears to quench fluorescence in some cells. Arrow indicates a WGA-HRP-labelled cell which appears as a hole in the tissue under UV light. B a r 50/~m.
observed in individual D R G cells in all 3 animals tested with double injections (Fig. 2, arrowheads). However, fewer than half of all labelled D R G cells were co-labelled. There appeared to be some interference between the two tracers as some cells containing H R P reaction product appeared as holes in the tissue when visualized under fluorescence (Fig. 2, arrow). The FG dehydration process resulted in a 38% decrease in the area of FG-labelled cell profiles. Prior to dehydration when mounted in glycerin or DMSO, 34 individually identified F G profiles measured 533 _+ 36 p~m2, whereas after dehydration via the FG protocol the same profiles measured 332_+ 24 ~zm2 (Fig. 3). There was no correlation between the initial profile area and the amount of shrinkage of each profile (Fig. 4). The cross-sectional areas of W G A - H R P cell profiles (processed via the H R P protocol) were not significantly different from the area of F G - l a b e l l e d / n o n - d e hydrated profiles. However, the average area of FGfilled profiles which underwent FG dehydration was significantly smaller than the FG labelled/non-dehydrated profiles. The FG cell profiles which underwent H R P dehydration (without undergoing the tetramethylbenzidine reaction process) were significantly smaller (300 _+ 2 6 / ~ m 2) than the other groups (Fig. 3). This study demonstrated that the difference in cross
sectional areas of bladder afferent neuronal profiles labelled with W G A - H R P and FG is due to post-fixation processing and dehydration and not to differential transport of the tracers in different populations of neurons. It was shown here that a given bladder sensory neuron can carry both FG and W G A - H R P , and that the observed 34% reduction in FG-labelled profile area as compared to W G A - H R P - l a b e l l e d profile area can be accounted for by the FG dehydration sequence. These results caution that when size comparisons are made between cells filled with different tracers, a careful analysis of the tissue processing methods must be
100
=
80O m
m [] []
[]
[] E m
n
D
tlm
[]
m
0
D
t~
m
tl la o
D
[]
m
[]
[]
m
~a
~0
1000
1200
Initial profile area Fig. 4. Amount of dehydration-induced shrinkage is unrelated to initial profile area. There was a wide variation in the amount of dehydration-induced shrinkage in FG-labelled profiles which was not correlated with the initial (pre-dehydration) profile area.
355 done to ensure that observed differences are not artifactual. The FG/WGA-HRP double-labelling studies showed that at least some of the bladder sensory neurons can carry both tracers. The failure of all of the neurons to exhibit double labelling is possibly due to a subpopulation of bladder neurons which only transport one tracer. However, we think it is more likely that the non-double-labelled neurons are capable of transporting both tracers, but do not show double labelling because of (1) non-overlapping or only partially overlapping injections of the two tracers, resulting in some singly labelled cells a n d / o r (2) quenching of F G fluorescence by W G A - H R P in some double-labelled cells 2 (see also Fig. 2). Thus, the actual number of doublelabelled neurons in this study may have been greater than what was observed, as heavily filled W G A - H R P cells appeared to interfere with the visualization of FG-labelled cells using fluorescence microscopy. An additional argument that all populations of bladder afferents (i.e. A~ and C-fibers) 9 can transport both F G and W G A - H R P is that the mean profile area of W G A - H R P cells (583 + 22 /zm 2) and non-dehydrated FG cells (533 + 36/xm 2) falls between the mean profile area of A~ (702 + 158 /zm 2) and C-fibers (449 + 47 /xm2) 4, which would indicate transport by both fiber groups. It may be a general rule that all peripheral afferents can transport both tracers as a recent study showed that both FG and W G A - H R P label all the sciatic nerve afferents in the rat, and therefore both are transported by all of the afferent subpopulations ( A a through C fibers) in the sciatic nerve 2. It should be noted that this other study applied the tracers to the cut sciatic nerve, therefore demonstrating transport by all fiber types, but not necessarily uptake by terminal endings. The FG-labelled cell profiles of the sciatic nerve were significantly smaller (26%) than the W G A H R P cells, which was attributed to dehydration-induced shrinkage 2. Other studies have shown that tissue dehydration does not necessarily give a fixed amount of shrinkage. It was shown that the amount of dehydration-induced brain shrinkage varied by brain region (44-59% shrinkage) 3 and thus a single correction factor for dehydration-induced shrinkage of all tissue types does not exist. It was also reported that ethanol dehydration in brain slices resulted in a shrinkage of Lucifer yellowfilled neurons to less than 2 / 3 of their original size, however, similar to the results obtained here, HRPfilled neurons did not exhibit shrinkage 3. The lack of shrinkage of HRP-filled cells is attributed to the formation of an insoluble precipitate during the H R P
reaction process which does not allow the cell to shrink when exposed to the H R P dehydration process 3. In contrast, it was shown here that FG-filled cells put through the H R P dehydration process actually shrank more than when put through the F G dehydration. This is presumably because the F G protocol used in our experiments had a more gradual change in ethanol concentration than the H R P protocol, resulting in smaller osmotic gradients and thus less tissue shrinkage 3. These results indicate that the method of tissue processing can lead to large artifactual changes in cell profile area. Thus a comparison of cell measurements from different studies must take into account methodology (e.g. ethanol dehydration sequence). Several recent studies have examined changes in cross-sectional area of specific neuronal populations due to various experimental perturbation 5'7'8. For example, urethral obstruction-induced hypertrophy of bladder post-ganglionic and D R G cells8; whereas spinal cord injury induced hypertrophy only of bladder D R G cells 5. It was demonstrated here that the initial profile area was unrelated to the degree of shrinkage of an individual cell indicating that large cells were not more apt to shrink than small ones. This indicates that the % change in neuronal cross-sectional area following a given experimental perturbation should be accurate even in dehydrated tissue. While it might be argued that DMSO or glycerinmounting of FG-labelled sections should routinely be done to avoid dehydration artifact, DMSO and glycerin mounting are less satisfactory than dehydration/Entellan mounting for long-term storage. However, if the absolute area of fluorescent dye-labelled cells is important, DMSO or glycerin mounting could be used to calculate a correction factor for that tissue, and if necessary, the coverslips then removed and the slides put through the normal FG dehydration process. In summary, all urinary bladder afferents appear to take up and transport both FG and W G A - H R P . However, the measured cross-sectional area of FG-labelled cell profiles was 34% less than that of W G A - H R P labelled profiles due to ethanol dehydration. This project was supported by a grant from the PVA Spinal Cord Research Foundation to M.N.K. and NIH Grant DK372421 to W.C.d.G. 1 Applebaum, A.E., Vance, W.H. and Coggeshall, R.E., Segmental localization of sensory cells that innervate the bladder, J. Comp. Neurol., 192 (1980) 203-209. 2 Baranowski, A.P., Anand, U. and McMahon, S.B., Retrograde labelling of dorsal root ganglion cells in the rat: a quantitative and morphological comparison of Fluoro-Gold with horseradish peroxidase labelling, Neurosci. Lett., 141 (1992) 53-56.
356 3 Grace, A.A. and Llinas, R., Morphological artifacts induced in intracellularly stained neurons by dehydration: circumvention using rapid dimethyl sulfoxide clearing, Neuroscience, 16 (1985) 461-475. 4 Harper, A.A. and Lawson, S.N., Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurones, Z Physiol., 359 (1985) 31-46. 5 Kruse, M.N., Erdman, S.L., Tanowitz, M. and de Groat, W.C., Differential effects of spinal cord injury on the morphology of bladder afferent and efferent neurons, Soc. Neurosci. Abstr., 18 (1992) 127. 6 Mesulam, W.M., The blue reaction product in horseradish peroxidase neurochemistry: incubation parameters and visibility, Z Histochem. Cytochem., 24 (1976) 1273-1280.
7 Steers, W.D., Ciambotti, J., Erdman, S. and de Groat, W.C., Morphological plasticity in efferent pathways to the urinary bladder of the rat following urethral obstruction, Z Neurosci., 10 (1990) 1943-1951. 8 Steers, W.D., Ciambotti, J., Etzel, B., Erdman, S. and de Groat, W.C., Alterations in afferent pathways from the urinary bladder of the rat in response to partial urethral obstruction, J. Comp. Neurol., 310 (1991) 401-410. 9 Vera, P.L. and Nadelhaft, I., Conduction velocity distribution of afferent fibers innervating the rat urinary bladder, Brain Res., 520 (1990) 83-89.