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A new method to quantify catecholamine stores visualized by means of the Falck-Hillarp technique
Neuroscience Letters, 10 (1978) 11--17 © Elsevier/North-Holland Scientific Publishers Ltd.
A NEW METHOD TO QUANTIFY CATECHOLAMINE BY MEANS OF THE FALCK-HILLARP TECHNIQUE
II
STORES VISUALIZED
L.F. AGNATI, F. BENFENATI, P. CORTELLI and R. D'ALESSANDRO Istituto di Fisiologia umana, Universita di Bologna, Piazza di Porta San Donato 2. 40127 Bologna (Italy) (ReceivedMay 1st,1978) (AcceptedJune 26th,1978)
SUMMARY
A new method has been developed by which it is possible to quantitate specific catecholamine fluorescence in Lhe CNS. The method is based on an elaboration by means of Kodalith plates of ::.iL Loph :*.3g=.ai=hs taken from ~uitable Falck-Hillarp preparations. This method il~LSbeen applied to study DA fmorescence decay in the caudatus after tyrosim, hydroxylase inhibition. The ,,cLhod is reliable since the half-life obtained for DA turnover in the caudatus is very close to similar values obtained by means .:ff both microfluorimetry and mass fragmentography.
The Falck-Hillarp iluore.-cence method ,~lows a histochemicM demonstration of monoamine stores, especially of catechol~xnines (CA) [ 2]. Since the fluorescence intensity of nerve terminals and cell b ~dies reflects CA contents, it is possible to evaluate CA stores by means of microfluorimeUic measurements of the fluorescence intensity of CA terminals vi.malized by means of the FalckHillarp technique [ 1,5,8,9]. With respect to biochemical dete .rminations this technique has the advantage that it allows the quanUtaUon of CA stores in discrete nerve terminal areas, without affecting the morphological features of th~ region. A parallel stu,.; : of the morpho!.ogy of CA terminals is therefore possible. The drawbacks of ,h~ ~ :rent quantitative a~,proach are the high cost ,~f a fluorescence microspec~.rograph equipped with a MPV system and the impossibility of an overall ~: aluation of a region (e.g. the median eminence). In fact this is possible by summing up and averaging a high number of measures obtained from single circular fields of small diameter (e.g. 13.5 urn, in refs. 8, 9). In contrast the present method is rather inexpensive. Furthermore, it allows to take into account very discrete fields and to give an overall measurement of the specific fluorescence of a whole region. Male Spl~gue-Dawley rats (150--200 g) were killed by rapid decapitation. The part of the brain to be processed was cut by means of a stereotaxic guillo-
12
r"
CA FLUORESCENCE INTENSITY
B
°I foe
/,
i
EXPOSURE TIME" ION KOO~LITH PLATE)
,
KODALITH PLATES
Fig. 1. Theoretical basis of the photographic elaboration allowing the separation of the continuous gray tone range of a microphotograph in equidensity areas. (A) On t~e x-axis, specific CA fluorescence intensities are reported. On the y-axis the corresponding u/~;cal densities (on Kodak Tri X Pan) are given; (B) The logarithms of expo~ure-time usir, Kodali~h plates and the corresponding density range are given on the x-axis and th~ !'axis, respectively. Since the contra~t of an emulsion is shown by the slope of the linear p ~ of the curve, the slope of the Kodalith one is very steep [see refs. 6 and 7); (C) On the xaxis three exposure-times (one for each Kodalith plate) are given. For each only gray :~.ones below a f'L~ed upper limit are revealed and within that range all the tones will appear irJ the Kodalith plate as equal gray tone (equidensity areas). Therefore 1;hree final positive Kodalith plate~ are obtained, one ('a') with high (H) plus middle (M) plus low (L) tones, a second one 'b' wPh high plus middle tones, and a third one ('c') with high tones only.
13
•
o
~[b
t
I
v
C
b
r
f
P 8
t
Fig. 2. Further elaboration on Kodalith plates after gray tone separation A d ~ k r o o m elaboration (see Fig. 1 ) has been carried out on a microphotograph taken from a fluorescence preparation of the NA terminals in the anterior hypothalamus (Kodak Tri-X Pan, magnification of x 250). Three Kodalith positive plates have been printed 'a', 'b', 'c'; Kodai ith plate 'a' shows L + M + H tones, 'b' shows M + H tones and plate 'c' shows H tones. An overall view of this tone separation can be seen in Fig. 2d (for further technical details see the text).
14
tine (allowing stereotaxic sections). Vibratome sections ( 2 0 - 3 0 p m thick) were made and then processed by a modification of the Falck-Hillarp technique which avoids perfusion (F. Benfenati et al., in prep.). Dopamine (DA) turnover w ~ evaluated by means of studying the Sl~.~ific DA fluorescence disappearance after tyrosine hydroxylase inhibition, which was obtained by a supramaximal dose (250 mg/kg i.p.) of H 44/68 (a-methyl-tyrosine methylester), (see ref. 2). The present method is based on darkroom elaborati,~n of the microphotographs obtahled by a fluorescence microscope (Zeiss IV) from suitable FalckHillarp preparations. A Kodak Tri X Pan film was used. Using high contrast Kodalith plates (for graphic : ~ , processed with homonymous bath, see refs. 6 and 7) it is possible to select gray intervals on the negatives (Fig. 1, A), by successive expouures (Fig. 1, B). Thus, it becomes possible to divide the set of tones in the microphotographs into discrete ranges (see Fig 1, C). Thus, the elaboration decomposes the original image into three equidensity areas (see Fig. 2(a--c)). The exposure times used in this elaboration were 5, 3.5, 2.5 see. To estimate the extent of equidensity areas it is enough to count the points included in the single areas on the three final positive images (area evaluation by the dot counting method, see ref. 4). The extent of the area corresponding to each of the three tone subdivisions (high tones H; middle tones M; low tones L) of the continuous range of gray tones in the original microphotograph, can be evaluated for the high tones frcm the area in the Kodalith plate 'c' (see Fig. 2c); for the middle tones by subtracting from the area shown in the Kidalith plate 'b' (see Fig. 2b) the area evaluated in the Kodalith plate 'c'; for the low tones by subtracting from the area shown in the Kodalith plate 'a' (see Fig. 2a) the one in the Kodalith plate 'b'. An overaU view can be obtained by printing the three Kodalith plates 'a' 'b' and 'c' in register on the same black and white or colour (using the additive system) photographic paper (see Fig. 2d). DA DISAPPEARANCE FROM RAT CAUDATUS AFTER TVROSINE HYDROXYLASE INHIBITION 200-
o
~J',-/~
!,/~=
101 m4n
~z 1 8 , 9 -
,,,z
~"- I EOi, >.
|
o
v
~
,2o
leo
x =., cm,nl
F~g. 3. Evaluation of DA turnover in the nucleus caudatus. On the x-axis, times after H44/6s t r e a t m e n t are given. On the y-axis the logarithm of the percentage ratio between the fluorescence intensity observed at time t (= 0', 90 °, 180') and the fluorescence intensity observed in the control group (t = 0 °) are reported. Each d o t represents the mean value (with its S.E.M.) of three observations. The best fit line (least square m e t h o d ) is given with its equation and the turnover rate is specified b y means of the half-line value.
•
-
•
i
,..
='~, .~..
..
-
o
. rl
41'
°
If"
o
t
lF •
.
--.-
.
,r
,.
: -. .. , : ,.. ... --~
. . . "
•
tl
i
i
I
'f
!
L4
Fig. 4. An example of the photographic elaboration, on which the D N turnover evaluation in Fig. 3, is based is reported in the three strips 0', 90', 180'. The original microphotographs were taken from the caudatus of animals killed 0, 90 and 180 rain after H44/~s injection. Each ~tr~p consists o f a separation o f gray tones in 4 ranges. The first column shows all the ;ones in each o f the three experimental situations (0, 90 and 180'). 'I~e last tone range (highest tones) for 90 and 180 rain was empty. Note that to simplify the dot-counti:lg procedure, each photo shows a grating made by dots, therefore it is enough to count directly the dots in each photo to get av evaluation of the respective area. (Magnification x 200).
180'
~0'
O,
r
t6 TABLE I COMPARISON OF DA T U R N O V E R EVALUATIONS IN CAUDATU8 Technique
HaSt.life tmin)
Soutt~
Mtcrofluorimetry
III
Mass fragmentography Photographic evslu .-.;~- .".
101
AEnoti et al., unpublished and ref. 1 Agnati et al., u n p u b l k h e d See Fig. 3, A
94
~mce tne darkness of the gray tone is a function of the CA fluorescence intensity, it is possible to evaluate such intensity in a fluorescent field by measuring the areas corresponding to H, L and M tones and then by adding the values a i Pi where ai is the area corresponding to the i th gray tone range in the positives, and Pi is a weight equal to the ~atural logarithm of the time exposttre used in the Kod~lith ~rocess to get that given i t'~ gray-tone range. In the tone separations illustrated in Figs. I and ~:, i = 1, 2, 3, that is the tone separation considers only three tone ranges. In the application of this method the t~)ne separation considers 4 gray tone ranl~es, to evaluate DA fluorescence inter sity in the, caudatus; the exposure times used in this elaboration were 5, 3.5, 2.b anc' 1.25 sec. Obviously, the more the gray tones ranges, the more precise is the fll~orescence intensity evaluation. The method has been applied to evaluate the dopamine contents decay m the caudatus after tyrosine hydroxylase inhibition (see Figs. 3 and 4). The resuits have been compared with similar evaluations carried out by using micr~fluorimetric and biochemical determinations of dopamine turnover in the nucleus caudatus (see Table I). It is worth noting that the evaluation of dopamine turnover obtained by this approach is intermediate to the ones of biochomleAI A_,~dmicrofluozimetric procedures. This technique avoids a major drawback of the microfluorimetric approach that is the virtual impossibility of evaluating CA stores in a l ~ g e re~cn t~ comparison with biochemical procedure, tl~e method has the advantage o," saving the morphological substrate and allowing measurements in very discrete brain areas. This technique can also be used to demonstrate and quantitatively evaluate (F. Benfenati et al., in prep.) islandic or dotted DA terminals [ 3] even if they are mixed with other diffusely fluorescent DA terminals since they have a higher intensity.
ACKNOWLEDGEMEN~
We thank Mr. G. Piancastelli (Zeiss Instruments) and Mr. G. Zoni for their skilfu! technical assistance.
17 REFERENCES 1 Einarsson, P., Hallman, B. and Jonsson, G., Quantitative microfluorimetry of formaldehyde-induced fluorescence of dopamine in caudate nucleus, Med. Biol., 53 (1975) 15--24. 2 Fuxe, K. and Jonsson, G., The histochemical fluorescence method for the demonstration of catecholamine. Theory, practice and application, J. Histochem. Cytochem., 21 (1973) 293--311. 3 Fuxe, K., II~kfeit, T., Goldstein, M., Johanuon and Agnati, L.F. Special morphological features of t,he neostriatal Dopamine innervation. In W. Birkmayer and O. Hornykiewicz (Eds.), Advances in Parkin~onism, Editions Roche, Basle, 1976, pp. 55--63. 4 Hennig, A., Fehlerbetrachtungen zur Volumenbestimung aus der Integration ebener Schnitte. In E.R. Weibel and H. Elias (~ds.), Quantitative methods in morphology, Springer, Berlin, 1967, pp. 99--129. 5 Jonsson, G., Quantitation of fluorescence of biogenic monoamines, Progr. Histochem. Cytochem., 2 (1971) 299--334. 6 Kodak Reports: Photographic material for the graphic arts., 1973. 7 Kodak Reports: Plates and films for science and industry, 1968. 8 L~fstr6m, A., Jonsson, G. alld Fuxe, R., Microfluorescence quantitation of catecholamine fluorescence in rat median eminence. I. Aspects on the distribution of dopamine and noradrenaline nerve terx.~inals, J. Histochem. Cytochem., 24 (1976) 414--429. 9 L~fstr6m, A., Jonsson, G., Wiesel, F.A. and Fuxe, R. Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. II. Turnover changes in hormonal states, J. Histochem. Cytochem., 24 (1976) 430--442.
A NEW METHOD TO QUANTIFY CATECHOLAMINE BY MEANS OF THE FALCK-HILLARP TECHNIQUE
II
STORES VISUALIZED
L.F. AGNATI, F. BENFENATI, P. CORTELLI and R. D'ALESSANDRO Istituto di Fisiologia umana, Universita di Bologna, Piazza di Porta San Donato 2. 40127 Bologna (Italy) (ReceivedMay 1st,1978) (AcceptedJune 26th,1978)
SUMMARY
A new method has been developed by which it is possible to quantitate specific catecholamine fluorescence in Lhe CNS. The method is based on an elaboration by means of Kodalith plates of ::.iL Loph :*.3g=.ai=hs taken from ~uitable Falck-Hillarp preparations. This method il~LSbeen applied to study DA fmorescence decay in the caudatus after tyrosim, hydroxylase inhibition. The ,,cLhod is reliable since the half-life obtained for DA turnover in the caudatus is very close to similar values obtained by means .:ff both microfluorimetry and mass fragmentography.
The Falck-Hillarp iluore.-cence method ,~lows a histochemicM demonstration of monoamine stores, especially of catechol~xnines (CA) [ 2]. Since the fluorescence intensity of nerve terminals and cell b ~dies reflects CA contents, it is possible to evaluate CA stores by means of microfluorimeUic measurements of the fluorescence intensity of CA terminals vi.malized by means of the FalckHillarp technique [ 1,5,8,9]. With respect to biochemical dete .rminations this technique has the advantage that it allows the quanUtaUon of CA stores in discrete nerve terminal areas, without affecting the morphological features of th~ region. A parallel stu,.; : of the morpho!.ogy of CA terminals is therefore possible. The drawbacks of ,h~ ~ :rent quantitative a~,proach are the high cost ,~f a fluorescence microspec~.rograph equipped with a MPV system and the impossibility of an overall ~: aluation of a region (e.g. the median eminence). In fact this is possible by summing up and averaging a high number of measures obtained from single circular fields of small diameter (e.g. 13.5 urn, in refs. 8, 9). In contrast the present method is rather inexpensive. Furthermore, it allows to take into account very discrete fields and to give an overall measurement of the specific fluorescence of a whole region. Male Spl~gue-Dawley rats (150--200 g) were killed by rapid decapitation. The part of the brain to be processed was cut by means of a stereotaxic guillo-
12
r"
CA FLUORESCENCE INTENSITY
B
°I foe
/,
i
EXPOSURE TIME" ION KOO~LITH PLATE)
,
KODALITH PLATES
Fig. 1. Theoretical basis of the photographic elaboration allowing the separation of the continuous gray tone range of a microphotograph in equidensity areas. (A) On t~e x-axis, specific CA fluorescence intensities are reported. On the y-axis the corresponding u/~;cal densities (on Kodak Tri X Pan) are given; (B) The logarithms of expo~ure-time usir, Kodali~h plates and the corresponding density range are given on the x-axis and th~ !'axis, respectively. Since the contra~t of an emulsion is shown by the slope of the linear p ~ of the curve, the slope of the Kodalith one is very steep [see refs. 6 and 7); (C) On the xaxis three exposure-times (one for each Kodalith plate) are given. For each only gray :~.ones below a f'L~ed upper limit are revealed and within that range all the tones will appear irJ the Kodalith plate as equal gray tone (equidensity areas). Therefore 1;hree final positive Kodalith plate~ are obtained, one ('a') with high (H) plus middle (M) plus low (L) tones, a second one 'b' wPh high plus middle tones, and a third one ('c') with high tones only.
13
•
o
~[b
t
I
v
C
b
r
f
P 8
t
Fig. 2. Further elaboration on Kodalith plates after gray tone separation A d ~ k r o o m elaboration (see Fig. 1 ) has been carried out on a microphotograph taken from a fluorescence preparation of the NA terminals in the anterior hypothalamus (Kodak Tri-X Pan, magnification of x 250). Three Kodalith positive plates have been printed 'a', 'b', 'c'; Kodai ith plate 'a' shows L + M + H tones, 'b' shows M + H tones and plate 'c' shows H tones. An overall view of this tone separation can be seen in Fig. 2d (for further technical details see the text).
14
tine (allowing stereotaxic sections). Vibratome sections ( 2 0 - 3 0 p m thick) were made and then processed by a modification of the Falck-Hillarp technique which avoids perfusion (F. Benfenati et al., in prep.). Dopamine (DA) turnover w ~ evaluated by means of studying the Sl~.~ific DA fluorescence disappearance after tyrosine hydroxylase inhibition, which was obtained by a supramaximal dose (250 mg/kg i.p.) of H 44/68 (a-methyl-tyrosine methylester), (see ref. 2). The present method is based on darkroom elaborati,~n of the microphotographs obtahled by a fluorescence microscope (Zeiss IV) from suitable FalckHillarp preparations. A Kodak Tri X Pan film was used. Using high contrast Kodalith plates (for graphic : ~ , processed with homonymous bath, see refs. 6 and 7) it is possible to select gray intervals on the negatives (Fig. 1, A), by successive expouures (Fig. 1, B). Thus, it becomes possible to divide the set of tones in the microphotographs into discrete ranges (see Fig 1, C). Thus, the elaboration decomposes the original image into three equidensity areas (see Fig. 2(a--c)). The exposure times used in this elaboration were 5, 3.5, 2.5 see. To estimate the extent of equidensity areas it is enough to count the points included in the single areas on the three final positive images (area evaluation by the dot counting method, see ref. 4). The extent of the area corresponding to each of the three tone subdivisions (high tones H; middle tones M; low tones L) of the continuous range of gray tones in the original microphotograph, can be evaluated for the high tones frcm the area in the Kodalith plate 'c' (see Fig. 2c); for the middle tones by subtracting from the area shown in the Kidalith plate 'b' (see Fig. 2b) the area evaluated in the Kodalith plate 'c'; for the low tones by subtracting from the area shown in the Kodalith plate 'a' (see Fig. 2a) the one in the Kodalith plate 'b'. An overaU view can be obtained by printing the three Kodalith plates 'a' 'b' and 'c' in register on the same black and white or colour (using the additive system) photographic paper (see Fig. 2d). DA DISAPPEARANCE FROM RAT CAUDATUS AFTER TVROSINE HYDROXYLASE INHIBITION 200-
o
~J',-/~
!,/~=
101 m4n
~z 1 8 , 9 -
,,,z
~"- I EOi, >.
|
o
v
~
,2o
leo
x =., cm,nl
F~g. 3. Evaluation of DA turnover in the nucleus caudatus. On the x-axis, times after H44/6s t r e a t m e n t are given. On the y-axis the logarithm of the percentage ratio between the fluorescence intensity observed at time t (= 0', 90 °, 180') and the fluorescence intensity observed in the control group (t = 0 °) are reported. Each d o t represents the mean value (with its S.E.M.) of three observations. The best fit line (least square m e t h o d ) is given with its equation and the turnover rate is specified b y means of the half-line value.
•
-
•
i
,..
='~, .~..
..
-
o
. rl
41'
°
If"
o
t
lF •
.
--.-
.
,r
,.
: -. .. , : ,.. ... --~
. . . "
•
tl
i
i
I
'f
!
L4
Fig. 4. An example of the photographic elaboration, on which the D N turnover evaluation in Fig. 3, is based is reported in the three strips 0', 90', 180'. The original microphotographs were taken from the caudatus of animals killed 0, 90 and 180 rain after H44/~s injection. Each ~tr~p consists o f a separation o f gray tones in 4 ranges. The first column shows all the ;ones in each o f the three experimental situations (0, 90 and 180'). 'I~e last tone range (highest tones) for 90 and 180 rain was empty. Note that to simplify the dot-counti:lg procedure, each photo shows a grating made by dots, therefore it is enough to count directly the dots in each photo to get av evaluation of the respective area. (Magnification x 200).
180'
~0'
O,
r
t6 TABLE I COMPARISON OF DA T U R N O V E R EVALUATIONS IN CAUDATU8 Technique
HaSt.life tmin)
Soutt~
Mtcrofluorimetry
III
Mass fragmentography Photographic evslu .-.;~- .".
101
AEnoti et al., unpublished and ref. 1 Agnati et al., u n p u b l k h e d See Fig. 3, A
94
~mce tne darkness of the gray tone is a function of the CA fluorescence intensity, it is possible to evaluate such intensity in a fluorescent field by measuring the areas corresponding to H, L and M tones and then by adding the values a i Pi where ai is the area corresponding to the i th gray tone range in the positives, and Pi is a weight equal to the ~atural logarithm of the time exposttre used in the Kod~lith ~rocess to get that given i t'~ gray-tone range. In the tone separations illustrated in Figs. I and ~:, i = 1, 2, 3, that is the tone separation considers only three tone ranges. In the application of this method the t~)ne separation considers 4 gray tone ranl~es, to evaluate DA fluorescence inter sity in the, caudatus; the exposure times used in this elaboration were 5, 3.5, 2.b anc' 1.25 sec. Obviously, the more the gray tones ranges, the more precise is the fll~orescence intensity evaluation. The method has been applied to evaluate the dopamine contents decay m the caudatus after tyrosine hydroxylase inhibition (see Figs. 3 and 4). The resuits have been compared with similar evaluations carried out by using micr~fluorimetric and biochemical determinations of dopamine turnover in the nucleus caudatus (see Table I). It is worth noting that the evaluation of dopamine turnover obtained by this approach is intermediate to the ones of biochomleAI A_,~dmicrofluozimetric procedures. This technique avoids a major drawback of the microfluorimetric approach that is the virtual impossibility of evaluating CA stores in a l ~ g e re~cn t~ comparison with biochemical procedure, tl~e method has the advantage o," saving the morphological substrate and allowing measurements in very discrete brain areas. This technique can also be used to demonstrate and quantitatively evaluate (F. Benfenati et al., in prep.) islandic or dotted DA terminals [ 3] even if they are mixed with other diffusely fluorescent DA terminals since they have a higher intensity.
ACKNOWLEDGEMEN~
We thank Mr. G. Piancastelli (Zeiss Instruments) and Mr. G. Zoni for their skilfu! technical assistance.
17 REFERENCES 1 Einarsson, P., Hallman, B. and Jonsson, G., Quantitative microfluorimetry of formaldehyde-induced fluorescence of dopamine in caudate nucleus, Med. Biol., 53 (1975) 15--24. 2 Fuxe, K. and Jonsson, G., The histochemical fluorescence method for the demonstration of catecholamine. Theory, practice and application, J. Histochem. Cytochem., 21 (1973) 293--311. 3 Fuxe, K., II~kfeit, T., Goldstein, M., Johanuon and Agnati, L.F. Special morphological features of t,he neostriatal Dopamine innervation. In W. Birkmayer and O. Hornykiewicz (Eds.), Advances in Parkin~onism, Editions Roche, Basle, 1976, pp. 55--63. 4 Hennig, A., Fehlerbetrachtungen zur Volumenbestimung aus der Integration ebener Schnitte. In E.R. Weibel and H. Elias (~ds.), Quantitative methods in morphology, Springer, Berlin, 1967, pp. 99--129. 5 Jonsson, G., Quantitation of fluorescence of biogenic monoamines, Progr. Histochem. Cytochem., 2 (1971) 299--334. 6 Kodak Reports: Photographic material for the graphic arts., 1973. 7 Kodak Reports: Plates and films for science and industry, 1968. 8 L~fstr6m, A., Jonsson, G. alld Fuxe, R., Microfluorescence quantitation of catecholamine fluorescence in rat median eminence. I. Aspects on the distribution of dopamine and noradrenaline nerve terx.~inals, J. Histochem. Cytochem., 24 (1976) 414--429. 9 L~fstr6m, A., Jonsson, G., Wiesel, F.A. and Fuxe, R. Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. II. Turnover changes in hormonal states, J. Histochem. Cytochem., 24 (1976) 430--442.