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On the use of the fluorescence histochemical method to estimate catecholamine content in brain
Neuropharmacoloyy. 1975. 14. 29t 299.PergamonPress.Printedin Gt. Britain.
ON THE USE OF THE FLUORESCENCE HISTOCHEMICAL METHOD TO ESTIMATE CATECHOLAMINE CONTENT IN BRAIN* N. G.
R. K. BHATNAGAR, W. J. SCHNUTEand L. S. VAN ORDEN Ill
BACOPOULOS,
The Toxicology Center, Department of Pharmacology. College of Medicine, University of Iowa, Iowa City, Iowa 52242 (Accepted 4 October 1974)
Summary--The relationship between intensity of formaldehyde-induced catecholamine fluorescence, determined microfluorometrically, and catecholamine content, measured biochemically, has been examined in various regions of rat brain. Linear relationships between these two parameters were obtained in the caudate nucleus, dorsomedial hypothalamus, arcuate nucleus, paraventricular nucleus, and internal and external layers of median eminence. Two pharmacological methods of catecholamine depletion were employed: inhibition of synthesis by ~-methyl-p-tyrosine and interference with storage by reserpine. Fluorescence intensity and catecholamine content declined in a proportionate manner following either drug. The catecholamine content of single median eminence specimens was measured by an enzymatic-isotopic method. Catecholamine content of larger brain parts was measured by a fluorometric method. Both analytical methods employed could differentiate between norepinephrine and dopamine. The histochemical method did not differentiate between the two amines. It is concluded that microfluorometric evaluation of histochemical fluorescence intensity can be a useful and valid estimate of catecholamine content in certain regions of the brain.
The formaldehyde vapour fluorescence histochemical method for the cellular demonstration of catecholamines (FALCK, 1962) has been employed in the investigation of a wide variety of peripheral and central catecholamine systems. The use of this technique for semiquantitative or quantitative measurements of peripheral catecholamines has been found to be subject to certain limitations. In autonomic nerves, fluorescence intensity determined microfluorometrically, and catecholamine content, determined by biochemical methods, were linearly related only when endogenous catecholamine content was lower than approximately 40~o of normal (VAN ORDEY, BENSCH,LANGERand TRENDELENBURG, 1967; JONSSON, 1969). This discrepancy has been explained in terms of fluorescence quenching caused by the high concentration of granule-bound catecholamines within granular vesicles in nerve terminals (RITZI~N, 1967). A recent report indicates that the number of fluorescent nerves measured by microfluorometric scanning correlates well with endogenous norepinephrine (NE) content in rat iris (SAcHs and JONSSON,1973). It is reasonable to enquire whether similar limitations may apply to the quantitative use of the histochemical fluorescence method in the central nervous system. In a study involving tissue smears from rat brain, fluorescence intensity changes in the hypothalamus could not be discerned by visual observation following as much as 500/odepletion of endogenous NE, whereas in the cortex a linear relationship was obtained between fluorescence estimated visually and NE content measured biochemically (L1DBRINKand JONSSON, 1971). Dopamine (DA) fluorescence in rat neostriatum determined by microfluorometry was found to decrease in proportion to endogenous DA content following administration of c~-methyl-p-tyrosine (c~-MPT) (FuXE and JONSSON,1972). It appears, therefore, that in the case of the hypothalamus, biochemical measurements concomitant with microfluorometric estimates are desirable whenever feasible. In spite of the uncertainty regarding the quantitative aspect of the histochemical method, it has occasionally been applied in a quantitative manner without parallel biochemical measurements. The turnover of catecholamines in the median eminence of rat * Supported in part by NIH Grants GM 12,675, Iowa Toxicology Center and GM 00141, Training in Pharmacology. 291
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hypothalamus has been estimated by visual observation of histochemical fluorescence intensity after treatment of animals with :¢-MPT (FuxE, HOKVELTand NILSSON, 1969; FUXE, H¢3KFELT,SUNDSTEDT,AHREN and HAMBERGER,1972). In a study of behavioural effects of ~-MPT, the histochemical method was used to quantitate the DA content of rat caudate nucleus microfluorometrically (DoWSON, 1973), but no concomitant biochemical data were provided. In a more recent study, changes in fluorescence intensity measured microfluorometrically were assumed to reflect changes in the DA content of tuberoinfundibular neuro n e s (LICHTENSTEIGER and KELLER, 1974). It would be desirable to obtain a precise delineation of the limits of reliability of the histochemical microfluorometric method as a quantitative procedure. The present study was undertaken to make a direct comparison between microfluorometric estimates and biochemical determinations of catecholamine content in various parts of rat brain. METHODS
Adult male rats (18(~200g) obtained from Hormone Assay Laboratories, kept on a regular light-dark cycle (12 hr dark, 12 light) and ad libitum feeding, were used throughout the study. Reserpine (Serpasil, Ciba) was injected intramuscularly in doses of 0.25, 0.50 or 1-00 mg/ kg, and ~-MPT methylester (Sigma) was injected in doses of 250 or 400 mg/kg intraperitoneally. Animals were sacrificed by decapitation. The brain was rapidly removed and placed on its dorsal surface. Two transverse sections were made, one at the level of the optic chiasm, the other at the rostral limit of the frontal poles to remove the attached portion of the olfactory bulbs. The tissue slice defined by the sections contained a major portion of the caudate nuclei. After disca~ing the septal and cortical parts of the slice, caudate specimens were used for biochemical or histochemical studies. The mean weight +_ S.E. of the dissected nuclei was 87.9 _+ 2.5 mg. A procedure similar to that described for the caudate was employed for the hypothalamus. Two transverse cuts at the level of the optic chiasm and mammillary bodies defined a tissue slice containing the hypothalamus. The slice was laid on its flat surface and two parasagittal sections were made at the lateral borders of the hypothalamus. The dorsal limit of the tissue specimen taken for analysis was defined by a section through the anterior commissure. The mean weight _+ S.E. of the hypothalamic specimens was 100.7 _+ 2.2 mg. The median eminence arcuate nucleus portion was removed from the hypothalamic specimen with the aid of a magnifying lens and fine scissors. The mean weight _+ S.E. of these specimens was 1.08 _+ 0.13 mg. Radioisotopic-enzymatic assay o/" NE and DA Norepinephrine and DA were analyzed according to the procedure described by COVLE and HENRY (1973). Briefly, each median eminence arcuate nucleus fragment was homogenized in 100 volumes of 0.1 M perchloric acid and centrifuged for 1 min at low speed. A maximum of 100/d of the supernatant was transferred to 15-ml glass-stoppered centrifuge tubes, to which the incubation mixture containing partially purified catechol-O-methyltransferase was added (for purification procedure see NIKODEJEViC, SENOH, DALY and CREVELING, 1970). The resultant mixture, which contained 140 #mol of Tris-HC1, pH 9.6, 0"5 #mol of MgC12, 780 nM of [3H]-S-adenosyl methionine (obtained from New England Nuclear, sp. act. = 8.02 Ci/mmol) and 3.24 /xmol of dithiothreitol, was incubated for 90 min at 37°C. The O-methylation reaction was stopped by addition of 150 /d of 1 M borate buffer, pH 11. At this point 7 /~g of methoxytyramine (Regis Chemical Company) and 3 #g of normetanephrine (Regis) were added to each sample. Ten ml of a watersaturated ethyl acetate-methanol (10:1) mixture were added to the samples, which were then mixed for 1 min and centrifuged for 4 min in an IEC centrifuge. Nine ml of the organic phase were mixed with 0.5 ml of 0.5 M borate buffer, pH 10 and centrifuged for 5 min. Eight ml of the resultant organic phase were transferred to a tube containing 0.5 ml of 0-1 M HC1, mixed for I min and centrifuged for 5 min. The organic
Catecholaminefluorescencehistochemistry
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phase was aspirated and the acid phase washed with 8 ml water-saturated ethyl acetate. The organic phase was discarded again and the tubes with the aqueous phase were transferred to an ice bath. Fifty ~1 of 3% sodium metaperiodate were added to each sample. Exactly 2 rain later, 50 #1 of 1~o glycerol were added to the samples. Ten ml of toluene were added, samples were mixed for 1 min and centrifuged for 10 min. Nine ml of the organic phase containing the methylated fragment of normetanephrine were transferred to a tube containing 1 ml of 1 MNaOH. The aqueous phase containing the [3H]-methoxytyramine was treated as described below (DA assay). NE assay. The toluene-NaOH mixture obtained above was mixed for 1 min and centrifuged for 10 rain. The organic phase was aspirated and 0-1 ml glacial acetic acid was added to the aqueous phase. After addition of l0 ml of toluene, samples were mixed for 1 min and centrifuged for 10 min. Nine ml of the organic phase were added to counting vials containing I0 ml of Aquasol (New England Nuclear) and counted in a model 3320 Packard Tri-Carb liquid scintillation spectrometer. DA assay. To the aqueous phase containing methoxytyramine, obtained as described above, 0.7ml of 1M borate buffer pH 11 was added to bring the final pH to 10, so that the methoxytyramine could be extracted. Six ml of a toluene-isoamyl alcohol solution (3:2) were added, and samples were mixed for I min and centrifuged for 10 rain. Four ml of the organic phase were transferred to counting vials containing 10 ml of Aquasol and counted. Blanks consisted of 100 #1 0.1 M perchloric acid. Three concentrations (2, 5 and 10 ng in 100/~1) of internal and external standards were used. In the case of the :~-MPT experiments, samples consisted of single median eminence specimens, whereas in the reserpine experiments two median eminence fragments were pooled. The unknown tissue values were calculated from the internal standards. Sensitivity of the assay was 100 pg for NE and 200 pg for DA. Twenty per cent of the NE appeared in the DA fraction due to incomplete cleavage of normetanephrine by sodium metaperiodate. Final values were corrected for this contamination. The assay did not differentiate between NE and epinephrine. Fluorometric assay of NE and DA
Specimens assayed for NE and DA content were homogenized in ice-cold perchloric acid, 0.4 M, containing 0'5 mg/ml ethylenediamine tetracetic acid (EDTA) and 0.5 mg/ml sodium metabisulphite. The homogenates were centrifuged at 20,000 g and potassium acetate, 10 M, was added to the supernatant to cause precipitation of excess perchlorate. The mixture was centrifuged again at 10,000 g and the catecholamines were obtained from the perchlorate-free supernatant by alumina adsorption at pH 8'0. Acetic acid, 0.1 M, was used to elute the catecholamines from the alumina. The eluate was centrifuged at 20,000 9 and the clear supernatant was brought to pH 6'5 by addition of an EDTA-sodium acetate solution and 0.2 M NaOH. The catecholamines in the mixture were then converted to their corresponding lutin fluorophores by iodine oxidation (CHANG, 1964). The final reaction products were stabilized by addition of 0'3 ml acetic acid, 5 M, which brought the solution to pH 5"8. Norepinephrine fluorescence was developed by heating the samples in a 100°C oven for 4 min (SHELLENBERGERand GORDON, 1971 ) and measured in an Aminco-Bowman spectroftuorometer at 410 mm excitation and 485 nm emission (uncorrected). The samples were then heated in a 100~C oven for an additional 35 min, cooled, and DA fluorescence was read at 300nm excitation and 390nm emission (uncorrected). Known amounts (between 10 and 200 ng) of standards were carried through the procedure in a manner identical to that for the samples and the unknown values were calculated from these standards. Recoveries were 72}o of NE and 70°~, of DA. H istochemical procedure Specimens taken for histochemical fluorescence measurements were prepared as follows: tissues were frozen in liquid nitrogen brought to near-freezing point by evacuation (L. J. ROTH, personal communication), then freeze-dried for 5-7 days over phosphorus pen+ toxide in a vacuum of 2-67 x 10 3 Pa, at -35°C. Following freeze-drying, the specimens were brought to room temperature and exposed to formaldehyde vapour at 80°C for 2 hr.
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The paraformaldehyde used was stored at 56~o relative humidity. The tissues were infiltrated with paraffin and 8 #m coronal sections were cut on a Leitz rotary microtome and mounted in mineral oil.
M icrospectrofluorometr y The sections were examined under a Leitz Orthoplan microscope equipped for microspectrofluorometry, modified from that described by VAN ORDEN (1970). Spectral analysis of the tissue sections was performed using a 1000 W Xenon lamp (Hanovia 976C-1) in a Schoeffel LH-151N lamp housing. The arc of the Xenon lamp was focused on the entrance slit of a motorized scanning monochromator (Schoeffel GM250). The exit image of the monochromator was focused onto the specimen by use of a Leitz immersion dark-field condenser. A fluorite oil 54 x 0.95 objective lens was used. Fluorescence emission from the specimen was transmitted through a Leitz microfluorometric attachment (MPV) to a Schoeffel GM100 motorized scanning monochromator and detected by an EMI 9658A photomultiplier tube, kept in a Schoeffel D500T thermoelectric cooled housing. The signal from the photomultiplier tube was amplified by a Schoeffel M600 photometer, the output of which was displayed on an Aminco X-Y recorder. M icr~?ltuorometry After the presence of catecholamine fluorophores had been verified by spectral analysis, the instrument was employed for microfluorometry. Excitation light from a 150W Osram Xenon lamp was transmitted to the specimen via excitation filters (Schott BG-3 and BG12) and BG-38 heat suppression filter and an E. Leitz Inc. incident light system (after Ploem) in the No. 3 position. The area of the specimen that was irradiated for each measurement was kept constant (200 #m2). The same photodetection system was used as for spectral analysis, but the output of the photometer was displayed on a Sanborn 7701-A strip recorder. A microfluorometric measurement consisted of a record of fluorescence emission intensity from a 100 ~m-" area from a given tissue section. Measurements of tissue autofluorescence and light scatter were obtained from non-catecholamine-containing areas and those values were subtracted from microfluorometric measurements from the areas under study (external and internal layers of the median eminence of the hypothalamus, periventricular and arcuate nuclei, dorsomedial hypothalamus and caudate nucleus).
Representation of histochemical data Three sections from the hypothalamus and three from the caudate nucleus of each animal were used for microfluorometric determinations. Ten measurements from a given area, such as arcuate nucleus, were obtained from each section. An average of 30 values obtained from a given animal constituted a histochemical fluorescence intensity measurement for a given brain area. Experimental groups consisted of four to six animals. The difference between any given histochemical fluorescence intensity value and the corresponding amine content was tested for statistical significance by one-way analysis of variance followed by the least significant difference multiple comparison procedure (STEELE and TORRIE, 1960). All values were converted to percent of control so that histochemical and biochemical results could be compared. RESULTS
Histochemical microfluorometric measurements were compared with determinations of catecholamine content in the same brain areas. Control values of catecholamine content were as follows: hypothalamic NE, 1-30 _+ 0'05 #g/g, DA, 0.49 _+ 0"02 #g/g; caudate nucleus DA, 8"86 _+ 1.0 #g/g; median eminence fragment NE, 3.42 + 0.30 #g/g, DA, 3.53 _+ 0-32 #g/g.
Caudate nucleus Histochemical fluorescence intensity and DA content declined in a proportional manner following treatment with either reserpine or ~-MPT. The extent of depletion was similar
Catecholamine fluorescencehistochemistry [
Dopamine in Coudote Nucleus
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295
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Fig. 1. Relationship between dopamine content and histochemical fluorescencein the caudate nucleus after treatment with reserpine. Animals were sacrificed 4 hr after injection of the doses indicated on the abscissa. Each point represents the mean ___S.E. of determinations from four animals.
Dopamine in Coudgte Nucleus tO0, ~ 8o ~
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Fig. 2. Relationshipbetween dopaminecontent and histochemical fluorescenceintensity in the caudate nucleus after treatment with ~-methyl-p-tyrosine(~-MPT). Dosesof the drug and time of sacrifice after injection are indicated on the abscissa. Each point represents the mean + S.E. of determinations from four animals.
following either drug (Figs. 1 and 2). The NE content of the nucleus is very low (COOPER, BLOOM and ROTH, 1970)and does not contribute significantly to catecholamine histochemical fluorescence.
Median eminenceJragment Microfluorometric measurements from the internal layer of the median eminence correlated well with NE content of the fragment, and microfluorometric measurements from the external layer correlated with DA content. The catecholamine stores of the median eminence were somewhat resistant to depletion by the doses of 2-MPT employed (Figs. 4 and 6), whereas they were more sensitive to reserpine (Figs. 3 and 5), in relation to other areas examined. Dopamine content and histochemical fluorescence intensity in the external layer were particularly sensitive to reserpine. Furthermore, DA content declined more rapidly than NE after treatment with either reserpine or ~.-MPT.
H ypothalamus Microfluorometric measurements from hypothalamic nuclei were compared with total catecholamine content (NE + DA) of hypothalamus. A poor relationship between fluorescence and content was observed after ~-MPT treatment (Fig. 8). Histochemical fluorescence and amine content were better correlated following reserpine depletion (Fig. 7). In most cases of the above experiments, fluorescence intensity was slightly or markedly higher than the corresponding catecholamine content when both values were compared as a percentage of control. It is known that certain treatment variables, such as relative humidity of the formaldehyde vapour and the time of exposure of the sample to the vapour, may affect the fluorescence yield (JoNSSON, 1969; RITZI~N, 1967). Treatment of the samples for one or two hours with paraformaldehyde stored at 56% relative humidity
296
N . G . BACOPOULOSet al. Dopomine in Medion Eminence 100 , H
80
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\\\
.~ 60 4o ~2o
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Reserpine (mg/kg)
Fig. 3. Relationship between dopamine content of the median eminence fragment and histochemical fluorescence intensity in the external layer of the median eminence 4 hr after treatment with reserpine. Histochemical results are represented as the mean _+ S.E. of determinations obtained from five or six animals. Biochemical results are represented as the mean _+ S.E. of determinations obtained from three samples of two pooled median eminence fragments.
yielded similar fluorescence intensities. Under our experimental conditions, use of paraformaldehyde stored under relative humidity of approximately 56% yielded high intensity of fluorescence with good visualization of axon terminals in the hypothalamus, and a linear fluorescence-content relationship in the median eminence and caudate nucleus. DISCUSSION
The validity of the formaldehyde fluorescence histochemical method as a quantitative procedure in the CNS has been investigated. Microfluorometric histochemical estimations in various brain areas were compared with fluorometric or enzymatic-isotopic assay determinations of catecholamine content of the same areas. Two pharmacological methods of depletion of catecholamines were employed: interference with storage by reserpine and inhibition of synthesis by ~-MPT. In the caudate nucleus a high positive correlation was observed between DA histochemical fluorescence measured microfluorometrically and DA content measured by a fluorometric assay. There are several reasons why caudate nucleus dopamine can be readily assayed by fluorometric techniques: the endogenous DA content is very high, interference by NE is low, and the mass of caudate tissue is large and easily dissected, as compared with other brain areas. Furthermore, the caudate nucleus appears to be composed of histochemically uniform tissue, since DA nerve terminals are evenly distributed throughout the nucleus. Two recent studies (ANDEN, FUXE, HAMBERGERand HO~ELT, 1966; DOWSOy, 1973)employed the formaldehyde histochemical fluorescence method to quantitate striatal DA, but no concomitant biochemical measurements were reported. Our results indicate that DA content and histochemical fluorescence are well correlated in the striatum. Dopamine in Median Eminence 100
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2o ithr 21hr 250 m(:J/kg 250 mg/kg Time Afler I~-Methyl Tyrosme
i 4hr 400 mg/kg
Fig. 4. Relationship between dopamine content of the median eminence fragment and histochemical fluorescence intensity in the external layer of the median eminence after treatment with 7-MPT. Histochemical results are represented as the mean _+ S.E. of determinations obtained from five or six animals. Biochemical results are represented as the mean + S.E. of determinations obtained from six animals.
Catecholamine fluorescence histochemistry
297
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Reserpine [mg/kg)
Fig. 5. Relationship between norepinephrine content of the median eminence fragment and histochemical fluorescence intensity in the internal layer of the median eminence 4 hr after treatment with reserpine. Histochemical results are represented as the mean + S.E. of determinations obtained from four or six animals. Biochemical results are represented as the mean _+ S.E. of determinations obtained from three samples of two pooled median eminence fragments.
Norepinephrine in Median Eminence
i
i00 eo
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~
Median Eminence
~ 4o
~ 2o 250 mglkg 250 rng/kg Time Afler a-Methyl Tyroslne
4C3 mglkq
Fig, 6. Relationshi p between norepinephrine content of the median eminence fragment and histochemical fluorescence intensity in the internal layer of the median eminence after treatment with • -MPT. Histochemical results are represented as the mean _ S.E. of determinations from five or six animals. Biochemical results are represented as the mean +_ S.E. of determinations from six animals.
Catecholamines in Hypothalamus I00,
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.
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~' 20
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Fig. 7. Relationship between total hypothalamic catecholamine content (NE + DA) and histochemical fluorescence intensity in paraventricular (PV), arcuate (ARC) and dorsomedial hypothalamic ( D M H ) nuclei, 4 hr after treatment with reserpine. Histochemical results are represented as the m e a n + S.E. of determinations obtained from five or six animals. Biochemical results are represented as the mean + S.E. of determinations from eight animals•
298
N.G. BACOPOULOSet al. Catecholamines In Hypothalamus H
~1
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~
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401
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4hr 400 mg/kg
Fig. 8. Relationship between total hypothalamic catecholamine content and histochemical fluorescence intensity in paraventricular, arcuate and dorsomediat hypothalamic nuclei, after treatment with ~-MPT. Histochemical results are represented as the mean _+ S.E. of determinations from five or six animals. Biochemical results are represented as the mean +_ S.E. of determinations obtained from eight animals. Significant differences are indicated by astemsks.
The results obtained from the comparison of quantitative histochemical and chemical analytical measurements in the hypothalamus were more complex than those obtained from the caudate nucleus. In an initial series of experiments, whole hypothalamic catecholamines were measured by a fluorometric method described above (Methods). Microfluorometric measurements from discrete hypothalamic areas were then compared with total hypothalamic catecholamine content. This comparison was based on the incorrect assumption that a relatively homogenous rate of depletion would be evidenced throughout the hypothalamus (BAcoPOULOS, SCHNUTE, BHATNAGARand VAN ORDEN, 1973). The microfluorometric data suggested that different sensitivities to depleting drugs were displayed by different hypothalamic nuclei following the same pharmacological treatment. Furthermore, in the external layer of the median eminence, wide discrepancies were observed between the decline of histochemical fluorescence and content (BAcoPOULOS et al., 1973). In order to resolve these questions isotopic-enzymatic measurements of NE and DA content of discrete hypothalamic areas were undertaken, since catecholamine content should be compared directly with micro-fluorometric histochemical estimations from the same area. The area of hypothalamus chosen for direct microfluorometric intensity-catecholamine content comparisons was the median eminence, because catecholaminergic systems in this structure have been studied extensively (FuxE et al., 1969, 1972). Furthermore, the greatest discrepancy between total hypothalamic catecholamine content and regional microfluorometric estimates was observed in the external layer of the median eminence following injection of reserpine or ~-MPT (BAcoPOULOSet al., 1973). The results shown in Figures 5 and 6 suggest a high positive correlation between the DA content measured by the enzymatic-isotopic method in the median eminence-arcuate nucleus fragment and microfluorometric measurements in the external layer of the median eminence. Our results pertaining to DA content of the median eminence fragment are in agreement with those of KAVANAGHand WEBZ (1974) who examined a similar piece of tissue using a fluorometric assay for catecholamines. Evidence fom other studies (BJORKLUND, MOORE, NOBIN and STENEVI, 1973; JONSSON, FUXE, and HOKFELT, 1972; HARTMAN, 1973; KAVANAGHand WEBZ, 1974) suggests that DA is the predominant catecholamine in the external layer of the median eminence. Norepinephrine levels in the median eminence-arcuate nucleus fragment appeared to correlate best with microfluorometric histochemical estimates in the internal layer of the median eminence. This finding is also in accordance with previous pharmacologic and morphologic studies (JoNSSON, FUXE and HOK~ELT, 1972). It should be noted that the tissue taken for enzymatic isotopic assay is not anatomically identical to the external layer of the median eminence from which microfluorometric histochemical estimates were obtained. The tissue fragment assayed can be presumed to contain some NE not included within the internal layer of the median eminence, and some DA not included within the external layer. It appears safe to assume, however, that most of the DA of the fragment assayed by the enzymati~isotopic method
Catecholamine fluorescence histochemistry
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is localized in the external layer of the median eminence, and is not present merely as a precursor of NE in noradrenergic terminals (HARTMAN,1973). The evidence presented in this report suggests that when appropriate comparisons are made between biochemically determined catecholamine content and intensity of formaldehyde-induced fluorescence, a high positive correlation between these two measurements obtains. A relationship between fluorescence and content should be firmly established for each brain region when quantitative microfluorometric histochemical investigations are undertaken. REFERENCES
AND~N, N-E., FUXE, K., HAMBERGER,B. and HOKFELT,T. (1966). A quantitative study on the nigrostriatal dopamine neuron system in the rat. Acta physiol, scand. 67: 306.
BACOPOULOS,N. G., SCHNUTE,W. J., BHATNAGAR,R. K. and VAN ORDEN, L. S. III (1973). Correlation of brain catecholamine content with microfluorometric histochemical determinations. Pharmacologist 15: 210.
BJORKLUND, A., MOORE. R. Y., NOBIN,A. and STENEVLU. (1973). The organization of tubero-hypothyseal and reticulo-infundibular catecholamine neuron systems in the rat brain. Brain Res. 51:171 191.
CHANG,C. C. (1964). A sensitive method for spectrophotofluorimetricassay of catecholamines. Int. J. Neuropharmac. 3:643 649.
COOPER, J. R., BLOOM,F. E. and ROTH, R. H. (1970). The Biochemical Basis of Neuropharmacology. p. 99. Oxford University Press, Oxford. COYLE, J. T. and HENRY, D. (1973). Catecholamines in fetal and newborn rat brain. J. Neurochem. 21: 61-67. DOWSON, J. H. (1973). Quantitative histochemical studies of striatal dopamine depletion following DL-~-methyl-ptyrosine administration. Neuropharmacology 12:949 953. FALCK, B. (1962). Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta physiol, scand. 56: Suppl., 197, 1. FUXE, K., HOKFELT,T. and NILSSON O. (1969). Castration, sex hormones, and tubero-infundibular dopamine neurons. Neuroendocrinology 5:107 120. FUXE, K., HOKFELT,T., SUNDSTEDT, C. D., AHREN, K. and HAMBERGER,L. (1972). Amine turnover changes in tubero-infundibular dopamine (DA)neurons in immature rats injected with PMS. Neuroendocrinology 10:282 300. FUXE, K. and JONSSON, G. (1972). The histochemical fluorescence method for the demonstration of catecholamines. J. Histochem. Cytochem. 21:293 311. HARTMAN, B. (1973). Immunofluorescence of dopamine fl-hydroxylase: application of improved methodology to the localization of the peripheral and central noradrenergic nervous system. J. Histochem. Cytochem. 21: 3 1 ~ 331. JONSSON, G. (1969). Microfluorometric studies on the formaldehyde-induced fluorescence of noradrenaline in adrenergic nerves of rat iris. J. Histochem. Cytochem. 17: 71~723. JONSSON, G., FUXE, K. and HOKFELT,T. (1972). On the catecholamine innervation of the hypothalamus, with special reference to the median eminence. Brain Res. 40:271 281. KAVANAGH,A. and WHsz, J. (1974). Localization of dopamine and norepinephrine in the medial basal hypothalamus of the rat. Neuroendoerinolo#y 13:201 212. LICHTENSTEIGER,W. and KELLER, P. J. (1974). Tubero-infundibular dopamine neurons and the secretion of luteinizing hormone and prolactin: Extrahypothalamic influences, interaction with cholinergic systems and the effect of urethane anesthesia. Brain Res. 74:279 303. LIDBRINK P. and JONSSON,G. (1971). Semiquantitative estimation of formaldehyde fluorescence of noradrenaline in central noradrenaline nerve terminals. J. Histochem. Cytochem. 198:747 757. NIKODEJEVIC,B., SENOH,S., DALY,J. W. and CREVEUNG, C. R. (1970). Catechol-O-methyltransferase. II. A new class of inhibitors of catechol-O-methyltransferase; 3,5,-dihydroxy-4-methoxybenzoicacid and related compounds. J. Pharmae. exp. Ther. 174: 83-93. RITZEN, M. (1967). Cytochemical identification and quantitation of biogenic monoamines, p. 39, M.D. Thesis, Karolinska Instituter, Stockholm. SAchs, C. and JONSSON,G. (1973). Quantitative microfluorometric and neurochemical studies on degenerating adrenergic nerves. J. Histochem. Cytochem. 21:902 911. SHELLENBERGER,M. K. and GORDON, J. H. (1971). A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine and 5-hydroxytryptamine from discrete brain areas. Analyt. Biochem. 39:. 356-373. STEELE,R. G. D. and TORRIE, J. H. (1960). Principles and Procedures of Statistics. p. 114. McGraw-Hill, New York. VAN ORDEN, L. S. III, BENSCH, K. G., LANGER,S. Z. and TRENDELENBURG,U. (1967). Histochemical and fine structural aspects of the onset of denervation supersensitivity in the nictitating membrane of the spinal cat. J. Pharmac. exp. Ther. 157: 274~282. VAN ORDEN, L. S. III (1970). Quantitative histochemistry of biogenic amines. A simple microspectrofluorometer. Biochem. Pharmac. 19:1105 1117.
ON THE USE OF THE FLUORESCENCE HISTOCHEMICAL METHOD TO ESTIMATE CATECHOLAMINE CONTENT IN BRAIN* N. G.
R. K. BHATNAGAR, W. J. SCHNUTEand L. S. VAN ORDEN Ill
BACOPOULOS,
The Toxicology Center, Department of Pharmacology. College of Medicine, University of Iowa, Iowa City, Iowa 52242 (Accepted 4 October 1974)
Summary--The relationship between intensity of formaldehyde-induced catecholamine fluorescence, determined microfluorometrically, and catecholamine content, measured biochemically, has been examined in various regions of rat brain. Linear relationships between these two parameters were obtained in the caudate nucleus, dorsomedial hypothalamus, arcuate nucleus, paraventricular nucleus, and internal and external layers of median eminence. Two pharmacological methods of catecholamine depletion were employed: inhibition of synthesis by ~-methyl-p-tyrosine and interference with storage by reserpine. Fluorescence intensity and catecholamine content declined in a proportionate manner following either drug. The catecholamine content of single median eminence specimens was measured by an enzymatic-isotopic method. Catecholamine content of larger brain parts was measured by a fluorometric method. Both analytical methods employed could differentiate between norepinephrine and dopamine. The histochemical method did not differentiate between the two amines. It is concluded that microfluorometric evaluation of histochemical fluorescence intensity can be a useful and valid estimate of catecholamine content in certain regions of the brain.
The formaldehyde vapour fluorescence histochemical method for the cellular demonstration of catecholamines (FALCK, 1962) has been employed in the investigation of a wide variety of peripheral and central catecholamine systems. The use of this technique for semiquantitative or quantitative measurements of peripheral catecholamines has been found to be subject to certain limitations. In autonomic nerves, fluorescence intensity determined microfluorometrically, and catecholamine content, determined by biochemical methods, were linearly related only when endogenous catecholamine content was lower than approximately 40~o of normal (VAN ORDEY, BENSCH,LANGERand TRENDELENBURG, 1967; JONSSON, 1969). This discrepancy has been explained in terms of fluorescence quenching caused by the high concentration of granule-bound catecholamines within granular vesicles in nerve terminals (RITZI~N, 1967). A recent report indicates that the number of fluorescent nerves measured by microfluorometric scanning correlates well with endogenous norepinephrine (NE) content in rat iris (SAcHs and JONSSON,1973). It is reasonable to enquire whether similar limitations may apply to the quantitative use of the histochemical fluorescence method in the central nervous system. In a study involving tissue smears from rat brain, fluorescence intensity changes in the hypothalamus could not be discerned by visual observation following as much as 500/odepletion of endogenous NE, whereas in the cortex a linear relationship was obtained between fluorescence estimated visually and NE content measured biochemically (L1DBRINKand JONSSON, 1971). Dopamine (DA) fluorescence in rat neostriatum determined by microfluorometry was found to decrease in proportion to endogenous DA content following administration of c~-methyl-p-tyrosine (c~-MPT) (FuXE and JONSSON,1972). It appears, therefore, that in the case of the hypothalamus, biochemical measurements concomitant with microfluorometric estimates are desirable whenever feasible. In spite of the uncertainty regarding the quantitative aspect of the histochemical method, it has occasionally been applied in a quantitative manner without parallel biochemical measurements. The turnover of catecholamines in the median eminence of rat * Supported in part by NIH Grants GM 12,675, Iowa Toxicology Center and GM 00141, Training in Pharmacology. 291
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hypothalamus has been estimated by visual observation of histochemical fluorescence intensity after treatment of animals with :¢-MPT (FuxE, HOKVELTand NILSSON, 1969; FUXE, H¢3KFELT,SUNDSTEDT,AHREN and HAMBERGER,1972). In a study of behavioural effects of ~-MPT, the histochemical method was used to quantitate the DA content of rat caudate nucleus microfluorometrically (DoWSON, 1973), but no concomitant biochemical data were provided. In a more recent study, changes in fluorescence intensity measured microfluorometrically were assumed to reflect changes in the DA content of tuberoinfundibular neuro n e s (LICHTENSTEIGER and KELLER, 1974). It would be desirable to obtain a precise delineation of the limits of reliability of the histochemical microfluorometric method as a quantitative procedure. The present study was undertaken to make a direct comparison between microfluorometric estimates and biochemical determinations of catecholamine content in various parts of rat brain. METHODS
Adult male rats (18(~200g) obtained from Hormone Assay Laboratories, kept on a regular light-dark cycle (12 hr dark, 12 light) and ad libitum feeding, were used throughout the study. Reserpine (Serpasil, Ciba) was injected intramuscularly in doses of 0.25, 0.50 or 1-00 mg/ kg, and ~-MPT methylester (Sigma) was injected in doses of 250 or 400 mg/kg intraperitoneally. Animals were sacrificed by decapitation. The brain was rapidly removed and placed on its dorsal surface. Two transverse sections were made, one at the level of the optic chiasm, the other at the rostral limit of the frontal poles to remove the attached portion of the olfactory bulbs. The tissue slice defined by the sections contained a major portion of the caudate nuclei. After disca~ing the septal and cortical parts of the slice, caudate specimens were used for biochemical or histochemical studies. The mean weight +_ S.E. of the dissected nuclei was 87.9 _+ 2.5 mg. A procedure similar to that described for the caudate was employed for the hypothalamus. Two transverse cuts at the level of the optic chiasm and mammillary bodies defined a tissue slice containing the hypothalamus. The slice was laid on its flat surface and two parasagittal sections were made at the lateral borders of the hypothalamus. The dorsal limit of the tissue specimen taken for analysis was defined by a section through the anterior commissure. The mean weight _+ S.E. of the hypothalamic specimens was 100.7 _+ 2.2 mg. The median eminence arcuate nucleus portion was removed from the hypothalamic specimen with the aid of a magnifying lens and fine scissors. The mean weight _+ S.E. of these specimens was 1.08 _+ 0.13 mg. Radioisotopic-enzymatic assay o/" NE and DA Norepinephrine and DA were analyzed according to the procedure described by COVLE and HENRY (1973). Briefly, each median eminence arcuate nucleus fragment was homogenized in 100 volumes of 0.1 M perchloric acid and centrifuged for 1 min at low speed. A maximum of 100/d of the supernatant was transferred to 15-ml glass-stoppered centrifuge tubes, to which the incubation mixture containing partially purified catechol-O-methyltransferase was added (for purification procedure see NIKODEJEViC, SENOH, DALY and CREVELING, 1970). The resultant mixture, which contained 140 #mol of Tris-HC1, pH 9.6, 0"5 #mol of MgC12, 780 nM of [3H]-S-adenosyl methionine (obtained from New England Nuclear, sp. act. = 8.02 Ci/mmol) and 3.24 /xmol of dithiothreitol, was incubated for 90 min at 37°C. The O-methylation reaction was stopped by addition of 150 /d of 1 M borate buffer, pH 11. At this point 7 /~g of methoxytyramine (Regis Chemical Company) and 3 #g of normetanephrine (Regis) were added to each sample. Ten ml of a watersaturated ethyl acetate-methanol (10:1) mixture were added to the samples, which were then mixed for 1 min and centrifuged for 4 min in an IEC centrifuge. Nine ml of the organic phase were mixed with 0.5 ml of 0.5 M borate buffer, pH 10 and centrifuged for 5 min. Eight ml of the resultant organic phase were transferred to a tube containing 0.5 ml of 0-1 M HC1, mixed for I min and centrifuged for 5 min. The organic
Catecholaminefluorescencehistochemistry
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phase was aspirated and the acid phase washed with 8 ml water-saturated ethyl acetate. The organic phase was discarded again and the tubes with the aqueous phase were transferred to an ice bath. Fifty ~1 of 3% sodium metaperiodate were added to each sample. Exactly 2 rain later, 50 #1 of 1~o glycerol were added to the samples. Ten ml of toluene were added, samples were mixed for 1 min and centrifuged for 10 min. Nine ml of the organic phase containing the methylated fragment of normetanephrine were transferred to a tube containing 1 ml of 1 MNaOH. The aqueous phase containing the [3H]-methoxytyramine was treated as described below (DA assay). NE assay. The toluene-NaOH mixture obtained above was mixed for 1 min and centrifuged for 10 rain. The organic phase was aspirated and 0-1 ml glacial acetic acid was added to the aqueous phase. After addition of l0 ml of toluene, samples were mixed for 1 min and centrifuged for 10 min. Nine ml of the organic phase were added to counting vials containing I0 ml of Aquasol (New England Nuclear) and counted in a model 3320 Packard Tri-Carb liquid scintillation spectrometer. DA assay. To the aqueous phase containing methoxytyramine, obtained as described above, 0.7ml of 1M borate buffer pH 11 was added to bring the final pH to 10, so that the methoxytyramine could be extracted. Six ml of a toluene-isoamyl alcohol solution (3:2) were added, and samples were mixed for I min and centrifuged for 10 rain. Four ml of the organic phase were transferred to counting vials containing 10 ml of Aquasol and counted. Blanks consisted of 100 #1 0.1 M perchloric acid. Three concentrations (2, 5 and 10 ng in 100/~1) of internal and external standards were used. In the case of the :~-MPT experiments, samples consisted of single median eminence specimens, whereas in the reserpine experiments two median eminence fragments were pooled. The unknown tissue values were calculated from the internal standards. Sensitivity of the assay was 100 pg for NE and 200 pg for DA. Twenty per cent of the NE appeared in the DA fraction due to incomplete cleavage of normetanephrine by sodium metaperiodate. Final values were corrected for this contamination. The assay did not differentiate between NE and epinephrine. Fluorometric assay of NE and DA
Specimens assayed for NE and DA content were homogenized in ice-cold perchloric acid, 0.4 M, containing 0'5 mg/ml ethylenediamine tetracetic acid (EDTA) and 0.5 mg/ml sodium metabisulphite. The homogenates were centrifuged at 20,000 g and potassium acetate, 10 M, was added to the supernatant to cause precipitation of excess perchlorate. The mixture was centrifuged again at 10,000 g and the catecholamines were obtained from the perchlorate-free supernatant by alumina adsorption at pH 8'0. Acetic acid, 0.1 M, was used to elute the catecholamines from the alumina. The eluate was centrifuged at 20,000 9 and the clear supernatant was brought to pH 6'5 by addition of an EDTA-sodium acetate solution and 0.2 M NaOH. The catecholamines in the mixture were then converted to their corresponding lutin fluorophores by iodine oxidation (CHANG, 1964). The final reaction products were stabilized by addition of 0'3 ml acetic acid, 5 M, which brought the solution to pH 5"8. Norepinephrine fluorescence was developed by heating the samples in a 100°C oven for 4 min (SHELLENBERGERand GORDON, 1971 ) and measured in an Aminco-Bowman spectroftuorometer at 410 mm excitation and 485 nm emission (uncorrected). The samples were then heated in a 100~C oven for an additional 35 min, cooled, and DA fluorescence was read at 300nm excitation and 390nm emission (uncorrected). Known amounts (between 10 and 200 ng) of standards were carried through the procedure in a manner identical to that for the samples and the unknown values were calculated from these standards. Recoveries were 72}o of NE and 70°~, of DA. H istochemical procedure Specimens taken for histochemical fluorescence measurements were prepared as follows: tissues were frozen in liquid nitrogen brought to near-freezing point by evacuation (L. J. ROTH, personal communication), then freeze-dried for 5-7 days over phosphorus pen+ toxide in a vacuum of 2-67 x 10 3 Pa, at -35°C. Following freeze-drying, the specimens were brought to room temperature and exposed to formaldehyde vapour at 80°C for 2 hr.
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The paraformaldehyde used was stored at 56~o relative humidity. The tissues were infiltrated with paraffin and 8 #m coronal sections were cut on a Leitz rotary microtome and mounted in mineral oil.
M icrospectrofluorometr y The sections were examined under a Leitz Orthoplan microscope equipped for microspectrofluorometry, modified from that described by VAN ORDEN (1970). Spectral analysis of the tissue sections was performed using a 1000 W Xenon lamp (Hanovia 976C-1) in a Schoeffel LH-151N lamp housing. The arc of the Xenon lamp was focused on the entrance slit of a motorized scanning monochromator (Schoeffel GM250). The exit image of the monochromator was focused onto the specimen by use of a Leitz immersion dark-field condenser. A fluorite oil 54 x 0.95 objective lens was used. Fluorescence emission from the specimen was transmitted through a Leitz microfluorometric attachment (MPV) to a Schoeffel GM100 motorized scanning monochromator and detected by an EMI 9658A photomultiplier tube, kept in a Schoeffel D500T thermoelectric cooled housing. The signal from the photomultiplier tube was amplified by a Schoeffel M600 photometer, the output of which was displayed on an Aminco X-Y recorder. M icr~?ltuorometry After the presence of catecholamine fluorophores had been verified by spectral analysis, the instrument was employed for microfluorometry. Excitation light from a 150W Osram Xenon lamp was transmitted to the specimen via excitation filters (Schott BG-3 and BG12) and BG-38 heat suppression filter and an E. Leitz Inc. incident light system (after Ploem) in the No. 3 position. The area of the specimen that was irradiated for each measurement was kept constant (200 #m2). The same photodetection system was used as for spectral analysis, but the output of the photometer was displayed on a Sanborn 7701-A strip recorder. A microfluorometric measurement consisted of a record of fluorescence emission intensity from a 100 ~m-" area from a given tissue section. Measurements of tissue autofluorescence and light scatter were obtained from non-catecholamine-containing areas and those values were subtracted from microfluorometric measurements from the areas under study (external and internal layers of the median eminence of the hypothalamus, periventricular and arcuate nuclei, dorsomedial hypothalamus and caudate nucleus).
Representation of histochemical data Three sections from the hypothalamus and three from the caudate nucleus of each animal were used for microfluorometric determinations. Ten measurements from a given area, such as arcuate nucleus, were obtained from each section. An average of 30 values obtained from a given animal constituted a histochemical fluorescence intensity measurement for a given brain area. Experimental groups consisted of four to six animals. The difference between any given histochemical fluorescence intensity value and the corresponding amine content was tested for statistical significance by one-way analysis of variance followed by the least significant difference multiple comparison procedure (STEELE and TORRIE, 1960). All values were converted to percent of control so that histochemical and biochemical results could be compared. RESULTS
Histochemical microfluorometric measurements were compared with determinations of catecholamine content in the same brain areas. Control values of catecholamine content were as follows: hypothalamic NE, 1-30 _+ 0'05 #g/g, DA, 0.49 _+ 0"02 #g/g; caudate nucleus DA, 8"86 _+ 1.0 #g/g; median eminence fragment NE, 3.42 + 0.30 #g/g, DA, 3.53 _+ 0-32 #g/g.
Caudate nucleus Histochemical fluorescence intensity and DA content declined in a proportional manner following treatment with either reserpine or ~-MPT. The extent of depletion was similar
Catecholamine fluorescencehistochemistry [
Dopamine in Coudote Nucleus
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295
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Fig. 1. Relationship between dopamine content and histochemical fluorescencein the caudate nucleus after treatment with reserpine. Animals were sacrificed 4 hr after injection of the doses indicated on the abscissa. Each point represents the mean ___S.E. of determinations from four animals.
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Fig. 2. Relationshipbetween dopaminecontent and histochemical fluorescenceintensity in the caudate nucleus after treatment with ~-methyl-p-tyrosine(~-MPT). Dosesof the drug and time of sacrifice after injection are indicated on the abscissa. Each point represents the mean + S.E. of determinations from four animals.
following either drug (Figs. 1 and 2). The NE content of the nucleus is very low (COOPER, BLOOM and ROTH, 1970)and does not contribute significantly to catecholamine histochemical fluorescence.
Median eminenceJragment Microfluorometric measurements from the internal layer of the median eminence correlated well with NE content of the fragment, and microfluorometric measurements from the external layer correlated with DA content. The catecholamine stores of the median eminence were somewhat resistant to depletion by the doses of 2-MPT employed (Figs. 4 and 6), whereas they were more sensitive to reserpine (Figs. 3 and 5), in relation to other areas examined. Dopamine content and histochemical fluorescence intensity in the external layer were particularly sensitive to reserpine. Furthermore, DA content declined more rapidly than NE after treatment with either reserpine or ~.-MPT.
H ypothalamus Microfluorometric measurements from hypothalamic nuclei were compared with total catecholamine content (NE + DA) of hypothalamus. A poor relationship between fluorescence and content was observed after ~-MPT treatment (Fig. 8). Histochemical fluorescence and amine content were better correlated following reserpine depletion (Fig. 7). In most cases of the above experiments, fluorescence intensity was slightly or markedly higher than the corresponding catecholamine content when both values were compared as a percentage of control. It is known that certain treatment variables, such as relative humidity of the formaldehyde vapour and the time of exposure of the sample to the vapour, may affect the fluorescence yield (JoNSSON, 1969; RITZI~N, 1967). Treatment of the samples for one or two hours with paraformaldehyde stored at 56% relative humidity
296
N . G . BACOPOULOSet al. Dopomine in Medion Eminence 100 , H
80
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.~ 60 4o ~2o
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Reserpine (mg/kg)
Fig. 3. Relationship between dopamine content of the median eminence fragment and histochemical fluorescence intensity in the external layer of the median eminence 4 hr after treatment with reserpine. Histochemical results are represented as the mean _+ S.E. of determinations obtained from five or six animals. Biochemical results are represented as the mean _+ S.E. of determinations obtained from three samples of two pooled median eminence fragments.
yielded similar fluorescence intensities. Under our experimental conditions, use of paraformaldehyde stored under relative humidity of approximately 56% yielded high intensity of fluorescence with good visualization of axon terminals in the hypothalamus, and a linear fluorescence-content relationship in the median eminence and caudate nucleus. DISCUSSION
The validity of the formaldehyde fluorescence histochemical method as a quantitative procedure in the CNS has been investigated. Microfluorometric histochemical estimations in various brain areas were compared with fluorometric or enzymatic-isotopic assay determinations of catecholamine content of the same areas. Two pharmacological methods of depletion of catecholamines were employed: interference with storage by reserpine and inhibition of synthesis by ~-MPT. In the caudate nucleus a high positive correlation was observed between DA histochemical fluorescence measured microfluorometrically and DA content measured by a fluorometric assay. There are several reasons why caudate nucleus dopamine can be readily assayed by fluorometric techniques: the endogenous DA content is very high, interference by NE is low, and the mass of caudate tissue is large and easily dissected, as compared with other brain areas. Furthermore, the caudate nucleus appears to be composed of histochemically uniform tissue, since DA nerve terminals are evenly distributed throughout the nucleus. Two recent studies (ANDEN, FUXE, HAMBERGERand HO~ELT, 1966; DOWSOy, 1973)employed the formaldehyde histochemical fluorescence method to quantitate striatal DA, but no concomitant biochemical measurements were reported. Our results indicate that DA content and histochemical fluorescence are well correlated in the striatum. Dopamine in Median Eminence 100
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i 4hr 400 mg/kg
Fig. 4. Relationship between dopamine content of the median eminence fragment and histochemical fluorescence intensity in the external layer of the median eminence after treatment with 7-MPT. Histochemical results are represented as the mean _+ S.E. of determinations obtained from five or six animals. Biochemical results are represented as the mean + S.E. of determinations obtained from six animals.
Catecholamine fluorescence histochemistry
297
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Reserpine [mg/kg)
Fig. 5. Relationship between norepinephrine content of the median eminence fragment and histochemical fluorescence intensity in the internal layer of the median eminence 4 hr after treatment with reserpine. Histochemical results are represented as the mean + S.E. of determinations obtained from four or six animals. Biochemical results are represented as the mean _+ S.E. of determinations obtained from three samples of two pooled median eminence fragments.
Norepinephrine in Median Eminence
i
i00 eo
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~
Median Eminence
~ 4o
~ 2o 250 mglkg 250 rng/kg Time Afler a-Methyl Tyroslne
4C3 mglkq
Fig, 6. Relationshi p between norepinephrine content of the median eminence fragment and histochemical fluorescence intensity in the internal layer of the median eminence after treatment with • -MPT. Histochemical results are represented as the mean _ S.E. of determinations from five or six animals. Biochemical results are represented as the mean +_ S.E. of determinations from six animals.
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~' 20
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Fig. 7. Relationship between total hypothalamic catecholamine content (NE + DA) and histochemical fluorescence intensity in paraventricular (PV), arcuate (ARC) and dorsomedial hypothalamic ( D M H ) nuclei, 4 hr after treatment with reserpine. Histochemical results are represented as the m e a n + S.E. of determinations obtained from five or six animals. Biochemical results are represented as the mean + S.E. of determinations from eight animals•
298
N.G. BACOPOULOSet al. Catecholamines In Hypothalamus H
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Fig. 8. Relationship between total hypothalamic catecholamine content and histochemical fluorescence intensity in paraventricular, arcuate and dorsomediat hypothalamic nuclei, after treatment with ~-MPT. Histochemical results are represented as the mean _+ S.E. of determinations from five or six animals. Biochemical results are represented as the mean +_ S.E. of determinations obtained from eight animals. Significant differences are indicated by astemsks.
The results obtained from the comparison of quantitative histochemical and chemical analytical measurements in the hypothalamus were more complex than those obtained from the caudate nucleus. In an initial series of experiments, whole hypothalamic catecholamines were measured by a fluorometric method described above (Methods). Microfluorometric measurements from discrete hypothalamic areas were then compared with total hypothalamic catecholamine content. This comparison was based on the incorrect assumption that a relatively homogenous rate of depletion would be evidenced throughout the hypothalamus (BAcoPOULOS, SCHNUTE, BHATNAGARand VAN ORDEN, 1973). The microfluorometric data suggested that different sensitivities to depleting drugs were displayed by different hypothalamic nuclei following the same pharmacological treatment. Furthermore, in the external layer of the median eminence, wide discrepancies were observed between the decline of histochemical fluorescence and content (BAcoPOULOS et al., 1973). In order to resolve these questions isotopic-enzymatic measurements of NE and DA content of discrete hypothalamic areas were undertaken, since catecholamine content should be compared directly with micro-fluorometric histochemical estimations from the same area. The area of hypothalamus chosen for direct microfluorometric intensity-catecholamine content comparisons was the median eminence, because catecholaminergic systems in this structure have been studied extensively (FuxE et al., 1969, 1972). Furthermore, the greatest discrepancy between total hypothalamic catecholamine content and regional microfluorometric estimates was observed in the external layer of the median eminence following injection of reserpine or ~-MPT (BAcoPOULOSet al., 1973). The results shown in Figures 5 and 6 suggest a high positive correlation between the DA content measured by the enzymatic-isotopic method in the median eminence-arcuate nucleus fragment and microfluorometric measurements in the external layer of the median eminence. Our results pertaining to DA content of the median eminence fragment are in agreement with those of KAVANAGHand WEBZ (1974) who examined a similar piece of tissue using a fluorometric assay for catecholamines. Evidence fom other studies (BJORKLUND, MOORE, NOBIN and STENEVI, 1973; JONSSON, FUXE, and HOKFELT, 1972; HARTMAN, 1973; KAVANAGHand WEBZ, 1974) suggests that DA is the predominant catecholamine in the external layer of the median eminence. Norepinephrine levels in the median eminence-arcuate nucleus fragment appeared to correlate best with microfluorometric histochemical estimates in the internal layer of the median eminence. This finding is also in accordance with previous pharmacologic and morphologic studies (JoNSSON, FUXE and HOK~ELT, 1972). It should be noted that the tissue taken for enzymatic isotopic assay is not anatomically identical to the external layer of the median eminence from which microfluorometric histochemical estimates were obtained. The tissue fragment assayed can be presumed to contain some NE not included within the internal layer of the median eminence, and some DA not included within the external layer. It appears safe to assume, however, that most of the DA of the fragment assayed by the enzymati~isotopic method
Catecholamine fluorescence histochemistry
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is localized in the external layer of the median eminence, and is not present merely as a precursor of NE in noradrenergic terminals (HARTMAN,1973). The evidence presented in this report suggests that when appropriate comparisons are made between biochemically determined catecholamine content and intensity of formaldehyde-induced fluorescence, a high positive correlation between these two measurements obtains. A relationship between fluorescence and content should be firmly established for each brain region when quantitative microfluorometric histochemical investigations are undertaken. REFERENCES
AND~N, N-E., FUXE, K., HAMBERGER,B. and HOKFELT,T. (1966). A quantitative study on the nigrostriatal dopamine neuron system in the rat. Acta physiol, scand. 67: 306.
BACOPOULOS,N. G., SCHNUTE,W. J., BHATNAGAR,R. K. and VAN ORDEN, L. S. III (1973). Correlation of brain catecholamine content with microfluorometric histochemical determinations. Pharmacologist 15: 210.
BJORKLUND, A., MOORE. R. Y., NOBIN,A. and STENEVLU. (1973). The organization of tubero-hypothyseal and reticulo-infundibular catecholamine neuron systems in the rat brain. Brain Res. 51:171 191.
CHANG,C. C. (1964). A sensitive method for spectrophotofluorimetricassay of catecholamines. Int. J. Neuropharmac. 3:643 649.
COOPER, J. R., BLOOM,F. E. and ROTH, R. H. (1970). The Biochemical Basis of Neuropharmacology. p. 99. Oxford University Press, Oxford. COYLE, J. T. and HENRY, D. (1973). Catecholamines in fetal and newborn rat brain. J. Neurochem. 21: 61-67. DOWSON, J. H. (1973). Quantitative histochemical studies of striatal dopamine depletion following DL-~-methyl-ptyrosine administration. Neuropharmacology 12:949 953. FALCK, B. (1962). Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta physiol, scand. 56: Suppl., 197, 1. FUXE, K., HOKFELT,T. and NILSSON O. (1969). Castration, sex hormones, and tubero-infundibular dopamine neurons. Neuroendocrinology 5:107 120. FUXE, K., HOKFELT,T., SUNDSTEDT, C. D., AHREN, K. and HAMBERGER,L. (1972). Amine turnover changes in tubero-infundibular dopamine (DA)neurons in immature rats injected with PMS. Neuroendocrinology 10:282 300. FUXE, K. and JONSSON, G. (1972). The histochemical fluorescence method for the demonstration of catecholamines. J. Histochem. Cytochem. 21:293 311. HARTMAN, B. (1973). Immunofluorescence of dopamine fl-hydroxylase: application of improved methodology to the localization of the peripheral and central noradrenergic nervous system. J. Histochem. Cytochem. 21: 3 1 ~ 331. JONSSON, G. (1969). Microfluorometric studies on the formaldehyde-induced fluorescence of noradrenaline in adrenergic nerves of rat iris. J. Histochem. Cytochem. 17: 71~723. JONSSON, G., FUXE, K. and HOKFELT,T. (1972). On the catecholamine innervation of the hypothalamus, with special reference to the median eminence. Brain Res. 40:271 281. KAVANAGH,A. and WHsz, J. (1974). Localization of dopamine and norepinephrine in the medial basal hypothalamus of the rat. Neuroendoerinolo#y 13:201 212. LICHTENSTEIGER,W. and KELLER, P. J. (1974). Tubero-infundibular dopamine neurons and the secretion of luteinizing hormone and prolactin: Extrahypothalamic influences, interaction with cholinergic systems and the effect of urethane anesthesia. Brain Res. 74:279 303. LIDBRINK P. and JONSSON,G. (1971). Semiquantitative estimation of formaldehyde fluorescence of noradrenaline in central noradrenaline nerve terminals. J. Histochem. Cytochem. 198:747 757. NIKODEJEVIC,B., SENOH,S., DALY,J. W. and CREVEUNG, C. R. (1970). Catechol-O-methyltransferase. II. A new class of inhibitors of catechol-O-methyltransferase; 3,5,-dihydroxy-4-methoxybenzoicacid and related compounds. J. Pharmae. exp. Ther. 174: 83-93. RITZEN, M. (1967). Cytochemical identification and quantitation of biogenic monoamines, p. 39, M.D. Thesis, Karolinska Instituter, Stockholm. SAchs, C. and JONSSON,G. (1973). Quantitative microfluorometric and neurochemical studies on degenerating adrenergic nerves. J. Histochem. Cytochem. 21:902 911. SHELLENBERGER,M. K. and GORDON, J. H. (1971). A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine and 5-hydroxytryptamine from discrete brain areas. Analyt. Biochem. 39:. 356-373. STEELE,R. G. D. and TORRIE, J. H. (1960). Principles and Procedures of Statistics. p. 114. McGraw-Hill, New York. VAN ORDEN, L. S. III, BENSCH, K. G., LANGER,S. Z. and TRENDELENBURG,U. (1967). Histochemical and fine structural aspects of the onset of denervation supersensitivity in the nictitating membrane of the spinal cat. J. Pharmac. exp. Ther. 157: 274~282. VAN ORDEN, L. S. III (1970). Quantitative histochemistry of biogenic amines. A simple microspectrofluorometer. Biochem. Pharmac. 19:1105 1117.