European Journal o f Pharmacology, 33 ( 1975) 125--130 © North-Holland Publishing C o m p a n y , A m s t e r d a m -- Printed in The Netherlands
AN A U T O R A D I O G R A P H I C METHOD OF MAPPING THE DISTRIBUTION AND DENSITY OF MONOAMINE N E U R O N S IN MOUSE BRAIN D A V I D T. M A S U O K A and A L E X F. A L C A R A Z
Neuropharmacology Research Lab., V.A. Hospital, Sepulveda, Calif. 91343, and Medical Pharmacology and Therapeutics, University of California, Irvine, Calif. 92664, U.S.A. Received 6 J a n u a r y 1975, revised MS received 17 April 1975, accepted 25 April 1975
D,T. M A S U O K A and A.F. A L C A R A Z , An auloradiographic melhod of mapping the distribution and density of monoamine neurons in mouse brain, European J. Pharmacol. 33 (1975) 125--130. A c o m b i n e d in vitro uptake and autoradiographic procedure as an i m p o r t a n t c o m p l e m e n t to the histochemical fluorescence m e t h o d is described. Slabs of fresh mouse brain were incubated with 14C_NE ' ~4C.DA or 14C.5.HT ' freeze-dried, and placed against X-ray film for autoradiography. Catecholamine nerve terminals were labeled by in vitro incubation with 14C.NE or 14C.DA" Dopaminergic terminals were labeled by 14C.NE incubation preceded by desipramine (to block uptake into NE terminals). With t 4C.5.HT incubation, the uptake pattern indicated the possibility that 5-HT nerve terminals were being labeled. Advantages of this m e t h o d are that it allows the visualization of overall density and distribution of selected m o n o a m i n e nerve terminals or uptake sites of o t h e r putative neurotransmitters in whole coronal or sagittal sections, so that data are o b t a i n e d from m a n y areas of brain or spinal cord rather than in only those areas preselected for microscopic viewing. Autoradiography
Norepinephrine
Dopamine
1. Introduction The development of the very sensitive histochemical fluorescence method for catecholamines (CA) and serotonin (5-HT) (Falck et al., 1962; Carlsson et al., 1962) has enabled detailed mapping of norepinephrine (NE), dopamine (DA) and 5-HT neuronal systems in the central nervous system (DahlstrSm and Fuxe, 1964; Fuxe, 1965; Ungerstedt, 1971}. The fluorescence method technically is a fairly difficult procedure and is limited in area to a small microscopic field unless a montage of several fields is produced. The latter procedure is quite time consuming. In addition, 5-HT nerve terminals are very fine and the fluorescence so labile that the system is difficult to map accurately. By means of histochemical fluorescence microscopy it has been demonstrated that NE
Serotonin
taken up by brain slices is accumulated into central CA neurons (Hamberger and Masuoka, 1965). Thus it appeared feasible, for the purpose of visualizing the extent and density of nerve terminals in neuroanatomically discrete areas of whole coronal sections of brain, to combine the in vitro uptake technique, which avoids the blood barrier, with autoradiography at a macro-level. This procedure was developed and, in fact, was applied during a study of 6-hydroxydopamine, a drug which causes the destruction of CA nerve terminals, to determine the extent of nerve terminal damage in the brain (Masuoka and Alcaraz, 1973) by labeling the surviving terminals. The present communication describes the details of the method and the distribution of labeling after incubation with 14C-labeled NE, DA and 5-HT. For the purpose of mapping the distribution and density of mono-
126
amine nerve terminals, this technique would be an important complement to the histochemical fluorescence method.
2. Materials and methods 48 male, white Swiss mice (Hilltop Lab Animals, Chatsworth, Calif.) weighing 20--25 g were used. The animals were decapitated and their brains quickly removed and placed in ice-cold Krebs bicarbonate medium (Krebs, 1950). Coronal slabs of mouse brain (less than one mm thick) were prepared by using a slicer consisting of 10 or 11 stainless steel doubleedged razor blades, each separated from each other by metal washers (about 0.8--0.9 mm thick), all held together by two nuts and screws. The chilled brain was placed on a cold paraffin block, and the pre-chilled slicer was quickly pushed through the brain. These brain slabs were placed in 10 ml of Krebs bicarbonate medium containing 0.1 pM ~4C-labeled NE, DA or 5-HT. 2-14C-DA hydrochloride (specific activity -- 57.3 mCi/mmol) and 2 -14C-5-HT creatinine sulfate (specific activity -- 57 mCi/mmol) were puchased from Amersham/ Searle Corp., Arlington Heights, Illinois, U.S.A. and 7-14 C-d,I-NE bitartrate (specific activity -44 mCi/mmol) from New England Nuclear, Boston, Massachusetts, U.S.A. Incubation was conducted with continuous shaking at 37 °C for 10 min under 95% 02 --5% CO2. After incubation, the tissue pieces were rinsed once with ice-cold Krebs medium and transferred to microscope slides. Excess medium was blotted with filter paper before placing the slabs against a microscope slide which was covered with teflon ('Plialine' teflon tape, 1 inch wide, The Lab Apparatus Co., Cleveland, Ohio, 44128, U.S.A.). This tape provided a smooth, flat, non-adhesive surface for the slabs during freezing horizontally in a freezer at --20°C. Before freeze-drying in vacuo over phosphorus pentoxide for 5--7 days, the brain slabs were peeled away easily from the teflon without cracking and revealing a smooth, flat, brain slab surface which is necessary to ensure intimate
D.T. MASUOKA, A.F. ALCARAZ
contact against the X-ray film (Gevaert Structurix) during exposure. Slabs were exposed for 7--9 weeks. For standardization all slabs from a particular experiment were exposed to film and developed simultaneously. Autoradiograms were compared against brain atlases of Montemurro and Dukelow (1972), Kovac and Denk (1968), and K6nig and Klippel (1963), and in selected instances slabs were stained with methyl green--pyronin.
3. Results Although more detailed localization studies are in progress in rats, the more obvious sites of radioactive distribution in slabs from 48 brains of mice are summarized below.
3.1. Catecholaminergic Incubating mouse brain slabs with 14C-NE resulted in uptake by catecholaminergic nerve terminals. There appeared to be no difference in the brain areas labeled whether 14 C-NE or ~4C_DA was used although some DA areas might have been labeled slightly higher with 14C_DA. Selected autoradiograms of brain slabs from various levels after 14C_NE incubation are illustrated in fig. lB. High levels of radioactivity were taken up by the nucleus accumbens, tuberculum olfactorium and nucleus interstitialis striae terminalis. In nucleus lateralis septi a diagonal line of high activity running ventrolaterally was observed. Medium levels were observed in the central and lateral parts of the amygdaloid complex. The hippocampus shows a low to medium level in the outer molecular layer and dentate gyms, no or very low activity in the radiatum, and a high line of activity in the outer layer. The exact localization is being studied in rats. In the basal ganglia, the nucleus caudatus/putamen showed very high uptake whereas the globus pallidus had no activity. The cerebral cortex showed a diffuse low to medium uptake of activity with a line of high radioactivity around the periphery of the cortex probably due to the presence of
AUTORADIOGRAPHIC MONOAMINE LOCALIZATION
CA nerve terminals (discussed below). A medium level was seen in the cingulum just adjacent to the corpus callosum. Certain areas of the diencephalon t o o k up high radioactivity. These included: nucleus paraventricularis rotundocellularis thalami, zona incerta and a portion of the medial forebrain bundle, nucleus periventricularis hypothalami, nucleus dorsomedialis hypothalami, an area bordering the base of the hypothalamus and the median eminence. Those areas taking up low to no activity included nucleus anterior hypothalami, nucleus ventromedialis hypothalami, fornix and medial lemniscus. The remainder of the thalamus, in general, showed low to medium uptake although the anterior ventral thalamus had slightly higher radioactivity. In the mesencephalon and lower brain stem there was a general low to medium uptake of radioactivity but medium to high levels in a portion of the substantia nigra, an area in and dorsal and lateral to nucleus interpeduncularis, a ventral part of the central gray and in the locus coeruleus area. A general low to medium level was observed in the cerebellum bordered by a high level of radioactivity.
3. 2. Dopaminergic In order to demonstrate dopaminergic neurons and terminals, mouse brain slabs were preincubated 5 min with desipramine (5 × 10 -7 M, DMI), an inhibitor of CA uptake (Hamberger, 1967), then ~4C-NE was added before incubation was continued for an additional 10 min. As can be seen in fig. 1A, the pretreatment with DMI drastically reduced uptake except in high DA-containing areas. Areas taking up very high levels of radioactivity (Fig. 1A) were nucleus caudatus/putamen, olfactory tubercle and median eminence. Medium to high levels were also observed in the nucleus lateralis septi, medial forebrain bundle, an area at the base of the hypothalamus lateral to the median eminence, a portion of the substantia nigra and an area lateral and dorso-
127
lateral to the nucleus interpeduncularis. There were low to medium levels in the central and lateral parts of the amygdaloid nuclei and some fibers in the internal capsule leading to nucleus caudatus/putamen.
3. 3. Serotoninergic C-5-HT was taken up by mouse brain slabs as shown in fig. 1C. There was a very high level of radioactivity around the periphery of the cerebral cortex (discussed below). The cerebral cortex showed a low to medium, diffuse level of uptake but with a definite line of higher activity lying midway and parallel to the surface. This layer is being studied in more detail in rats. A medium level of activity was observed in the nucleus caudatus/putamen, globus pallidus, nucleus accumbens, ~ b e r c u l u m olfactorium, amygdaloid complex and cingulum. The hippocampus showed a medium level of uptake in the outer molecular layer and inner radiatum. A layer relatively free of activity was observed in possibly the granular layer but this is also being studied in detail. There was high activity in and around the suprachiasmatic nucleus and in the lateral ventricles probably associated with the choroid plexus. Slightly higher levels were found in most of the hypothalamus, especially the dorsomedial and lateral areas. The median eminence also t o o k up ~4C-5-HT. The anterior, dorsal, lateral and medial thalami had medium activity but the ventral thalamus appeared to be relatively free of activity. Nucleus medialis habenulae was highly labeled while nucleus habenulae lateralis remained relatively unlabeled. The corpus callosum, fimbria hippocampi, internal capsule, optic tract, mammilothalamic tract and fornix also had low to no uptake of radioactivity. There appeared to be a general, medium level of uptake in the mesencephalon and lower brain stem with higher levels in the substantia nigra, interpeduncular nucleus and areas dorsolateral to the latter nucleus. Slightly higher 4
A
B
C
a
b
C
d
e
Fig. 1Aa--e. DA areas in mouse brain labeled by autoradiographic localization of 14C.NE uptake in the presence of DMI in vitro. Light areas represent radioactivity. The brain slabs are taken from approximately the telencephaIon near the anterior commissure (a), the anterior hypothalamus (b), the medial hypothalamus (c), the mesencephalon (d) and the metencephalon (e). Fig. 1Ba--e. CA areas (including NE and DA) in mouse brain labeled by autoradiographie localization of 14C-NE uptake in vitro. Fig. 1Ca--e. 5-HT areas in mouse brain labeled by autoradiographic localization of 14C-5-HT uptake in vitro. Key: 1, cerebral cortex; 2, nucleus caudatus/putamen; 3, septum; 4, hippocampus; 5, nucleus ventralis thalami; 6, globus pallidus; 7, median eminence; 8, nucleus ventromedialis hypothalami; 9, nucleus amygdaloideus; 10, cerebellum.
AUTORADIOGRAPHIC MONOAMINE LOCALIZATION
levels w e r e also o b s e r v e d in t h e central gray and
raphe. T h e cerebellar c o r t e x s h o w e d a very l o w level o f ~ 4 C-5-HT u p t a k e .
4. D i s c u s s i o n I n c u b a t i o n of coronal brain slabs with ~4 CNE resulted in an ubiquitous labeling of all CA neurons (fig. 1B) including NE, DA and possibly the newly described adrenaline neurons (H6kfelt et al., 1974b). The fact that 14 C-NE is t a k e n up into brain areas to a level roughly paralleling the density of fluorescent nerve terminals and tracts as demonstrated by the Falck--Hillarp technique (Dahlstr6m and Fuxe, 1964; Fuxe, 1965; Ungerstedt, 1971), that uptake is lacking after intraventricular 6-hydroxydopamine pretreatment (Masuoka and Alcaraz, 1973} and is inhibited after desipramine indicates that specific uptake into catecholaminergic neurons was involved. The diagonal line of terminals running ventrolaterally in the nucleus lateralis septi was identified previously as being catecholaminergic by autoradiography (Masuoka and Alcaraz, 1973) and by histofluorescence (Jacobowitz and Palkovits, 1974). With the use of DMI to block the uptake of CA into NE terminals b u t not DA terminals, this particular pattern of distribution appears in the autoradiogram to be dopaminergic. Since in a recent histochemical fluorescence study utilizing a combination of drug pretreatments (Lidbrink et al., 1974), the authors concluded that 'some of the CA terminals in the septal area may be DA nerve terminals', the present autoradiographic result provides further support for the view that these terminals indeed are dopaminergic. Other areas such as the corpus striatum observed previously in the fluorescence microscope as DA areas in the rat have been accurately labeled. The recently described DA terminals in the cerebral cortex (Thierry et al., 1973; H6kfelt et al., 1974a) were not readily apparent in these mouse slabs. At this point, it appears that the fine DA terminals in the cortex are not distin-
129
guishable with certainty by this method. The high 14 C-NE activity around the periphery or superficial layers of the cerebral cortex (Ba-e) has been discussed in detail previously (Masuoka and Alcaraz, 1973). It was concluded that high radioactivity was due to uptake into CA nerve terminals possibly in the pia or outer cortical layers (Falck et al., 1965; Fuxe et al., 1968a). The possibility of an artifact is excluded since brain slabs incubated in the absence of radioactivity and otherwise processed identically produced no traces on the X-ray film. Furthermore, after DMI pretreatment this ring of high activity, although not completely absent, was markedly reduced (Aa-e) strongly suggesting the presence of NE nerve terminals. The high 14 C-5-HT activity around the periphery of the cerebral cortex (Ca
AN A U T O R A D I O G R A P H I C METHOD OF MAPPING THE DISTRIBUTION AND DENSITY OF MONOAMINE N E U R O N S IN MOUSE BRAIN D A V I D T. M A S U O K A and A L E X F. A L C A R A Z
Neuropharmacology Research Lab., V.A. Hospital, Sepulveda, Calif. 91343, and Medical Pharmacology and Therapeutics, University of California, Irvine, Calif. 92664, U.S.A. Received 6 J a n u a r y 1975, revised MS received 17 April 1975, accepted 25 April 1975
D,T. M A S U O K A and A.F. A L C A R A Z , An auloradiographic melhod of mapping the distribution and density of monoamine neurons in mouse brain, European J. Pharmacol. 33 (1975) 125--130. A c o m b i n e d in vitro uptake and autoradiographic procedure as an i m p o r t a n t c o m p l e m e n t to the histochemical fluorescence m e t h o d is described. Slabs of fresh mouse brain were incubated with 14C_NE ' ~4C.DA or 14C.5.HT ' freeze-dried, and placed against X-ray film for autoradiography. Catecholamine nerve terminals were labeled by in vitro incubation with 14C.NE or 14C.DA" Dopaminergic terminals were labeled by 14C.NE incubation preceded by desipramine (to block uptake into NE terminals). With t 4C.5.HT incubation, the uptake pattern indicated the possibility that 5-HT nerve terminals were being labeled. Advantages of this m e t h o d are that it allows the visualization of overall density and distribution of selected m o n o a m i n e nerve terminals or uptake sites of o t h e r putative neurotransmitters in whole coronal or sagittal sections, so that data are o b t a i n e d from m a n y areas of brain or spinal cord rather than in only those areas preselected for microscopic viewing. Autoradiography
Norepinephrine
Dopamine
1. Introduction The development of the very sensitive histochemical fluorescence method for catecholamines (CA) and serotonin (5-HT) (Falck et al., 1962; Carlsson et al., 1962) has enabled detailed mapping of norepinephrine (NE), dopamine (DA) and 5-HT neuronal systems in the central nervous system (DahlstrSm and Fuxe, 1964; Fuxe, 1965; Ungerstedt, 1971}. The fluorescence method technically is a fairly difficult procedure and is limited in area to a small microscopic field unless a montage of several fields is produced. The latter procedure is quite time consuming. In addition, 5-HT nerve terminals are very fine and the fluorescence so labile that the system is difficult to map accurately. By means of histochemical fluorescence microscopy it has been demonstrated that NE
Serotonin
taken up by brain slices is accumulated into central CA neurons (Hamberger and Masuoka, 1965). Thus it appeared feasible, for the purpose of visualizing the extent and density of nerve terminals in neuroanatomically discrete areas of whole coronal sections of brain, to combine the in vitro uptake technique, which avoids the blood barrier, with autoradiography at a macro-level. This procedure was developed and, in fact, was applied during a study of 6-hydroxydopamine, a drug which causes the destruction of CA nerve terminals, to determine the extent of nerve terminal damage in the brain (Masuoka and Alcaraz, 1973) by labeling the surviving terminals. The present communication describes the details of the method and the distribution of labeling after incubation with 14C-labeled NE, DA and 5-HT. For the purpose of mapping the distribution and density of mono-
126
amine nerve terminals, this technique would be an important complement to the histochemical fluorescence method.
2. Materials and methods 48 male, white Swiss mice (Hilltop Lab Animals, Chatsworth, Calif.) weighing 20--25 g were used. The animals were decapitated and their brains quickly removed and placed in ice-cold Krebs bicarbonate medium (Krebs, 1950). Coronal slabs of mouse brain (less than one mm thick) were prepared by using a slicer consisting of 10 or 11 stainless steel doubleedged razor blades, each separated from each other by metal washers (about 0.8--0.9 mm thick), all held together by two nuts and screws. The chilled brain was placed on a cold paraffin block, and the pre-chilled slicer was quickly pushed through the brain. These brain slabs were placed in 10 ml of Krebs bicarbonate medium containing 0.1 pM ~4C-labeled NE, DA or 5-HT. 2-14C-DA hydrochloride (specific activity -- 57.3 mCi/mmol) and 2 -14C-5-HT creatinine sulfate (specific activity -- 57 mCi/mmol) were puchased from Amersham/ Searle Corp., Arlington Heights, Illinois, U.S.A. and 7-14 C-d,I-NE bitartrate (specific activity -44 mCi/mmol) from New England Nuclear, Boston, Massachusetts, U.S.A. Incubation was conducted with continuous shaking at 37 °C for 10 min under 95% 02 --5% CO2. After incubation, the tissue pieces were rinsed once with ice-cold Krebs medium and transferred to microscope slides. Excess medium was blotted with filter paper before placing the slabs against a microscope slide which was covered with teflon ('Plialine' teflon tape, 1 inch wide, The Lab Apparatus Co., Cleveland, Ohio, 44128, U.S.A.). This tape provided a smooth, flat, non-adhesive surface for the slabs during freezing horizontally in a freezer at --20°C. Before freeze-drying in vacuo over phosphorus pentoxide for 5--7 days, the brain slabs were peeled away easily from the teflon without cracking and revealing a smooth, flat, brain slab surface which is necessary to ensure intimate
D.T. MASUOKA, A.F. ALCARAZ
contact against the X-ray film (Gevaert Structurix) during exposure. Slabs were exposed for 7--9 weeks. For standardization all slabs from a particular experiment were exposed to film and developed simultaneously. Autoradiograms were compared against brain atlases of Montemurro and Dukelow (1972), Kovac and Denk (1968), and K6nig and Klippel (1963), and in selected instances slabs were stained with methyl green--pyronin.
3. Results Although more detailed localization studies are in progress in rats, the more obvious sites of radioactive distribution in slabs from 48 brains of mice are summarized below.
3.1. Catecholaminergic Incubating mouse brain slabs with 14C-NE resulted in uptake by catecholaminergic nerve terminals. There appeared to be no difference in the brain areas labeled whether 14 C-NE or ~4C_DA was used although some DA areas might have been labeled slightly higher with 14C_DA. Selected autoradiograms of brain slabs from various levels after 14C_NE incubation are illustrated in fig. lB. High levels of radioactivity were taken up by the nucleus accumbens, tuberculum olfactorium and nucleus interstitialis striae terminalis. In nucleus lateralis septi a diagonal line of high activity running ventrolaterally was observed. Medium levels were observed in the central and lateral parts of the amygdaloid complex. The hippocampus shows a low to medium level in the outer molecular layer and dentate gyms, no or very low activity in the radiatum, and a high line of activity in the outer layer. The exact localization is being studied in rats. In the basal ganglia, the nucleus caudatus/putamen showed very high uptake whereas the globus pallidus had no activity. The cerebral cortex showed a diffuse low to medium uptake of activity with a line of high radioactivity around the periphery of the cortex probably due to the presence of
AUTORADIOGRAPHIC MONOAMINE LOCALIZATION
CA nerve terminals (discussed below). A medium level was seen in the cingulum just adjacent to the corpus callosum. Certain areas of the diencephalon t o o k up high radioactivity. These included: nucleus paraventricularis rotundocellularis thalami, zona incerta and a portion of the medial forebrain bundle, nucleus periventricularis hypothalami, nucleus dorsomedialis hypothalami, an area bordering the base of the hypothalamus and the median eminence. Those areas taking up low to no activity included nucleus anterior hypothalami, nucleus ventromedialis hypothalami, fornix and medial lemniscus. The remainder of the thalamus, in general, showed low to medium uptake although the anterior ventral thalamus had slightly higher radioactivity. In the mesencephalon and lower brain stem there was a general low to medium uptake of radioactivity but medium to high levels in a portion of the substantia nigra, an area in and dorsal and lateral to nucleus interpeduncularis, a ventral part of the central gray and in the locus coeruleus area. A general low to medium level was observed in the cerebellum bordered by a high level of radioactivity.
3. 2. Dopaminergic In order to demonstrate dopaminergic neurons and terminals, mouse brain slabs were preincubated 5 min with desipramine (5 × 10 -7 M, DMI), an inhibitor of CA uptake (Hamberger, 1967), then ~4C-NE was added before incubation was continued for an additional 10 min. As can be seen in fig. 1A, the pretreatment with DMI drastically reduced uptake except in high DA-containing areas. Areas taking up very high levels of radioactivity (Fig. 1A) were nucleus caudatus/putamen, olfactory tubercle and median eminence. Medium to high levels were also observed in the nucleus lateralis septi, medial forebrain bundle, an area at the base of the hypothalamus lateral to the median eminence, a portion of the substantia nigra and an area lateral and dorso-
127
lateral to the nucleus interpeduncularis. There were low to medium levels in the central and lateral parts of the amygdaloid nuclei and some fibers in the internal capsule leading to nucleus caudatus/putamen.
3. 3. Serotoninergic C-5-HT was taken up by mouse brain slabs as shown in fig. 1C. There was a very high level of radioactivity around the periphery of the cerebral cortex (discussed below). The cerebral cortex showed a low to medium, diffuse level of uptake but with a definite line of higher activity lying midway and parallel to the surface. This layer is being studied in more detail in rats. A medium level of activity was observed in the nucleus caudatus/putamen, globus pallidus, nucleus accumbens, ~ b e r c u l u m olfactorium, amygdaloid complex and cingulum. The hippocampus showed a medium level of uptake in the outer molecular layer and inner radiatum. A layer relatively free of activity was observed in possibly the granular layer but this is also being studied in detail. There was high activity in and around the suprachiasmatic nucleus and in the lateral ventricles probably associated with the choroid plexus. Slightly higher levels were found in most of the hypothalamus, especially the dorsomedial and lateral areas. The median eminence also t o o k up ~4C-5-HT. The anterior, dorsal, lateral and medial thalami had medium activity but the ventral thalamus appeared to be relatively free of activity. Nucleus medialis habenulae was highly labeled while nucleus habenulae lateralis remained relatively unlabeled. The corpus callosum, fimbria hippocampi, internal capsule, optic tract, mammilothalamic tract and fornix also had low to no uptake of radioactivity. There appeared to be a general, medium level of uptake in the mesencephalon and lower brain stem with higher levels in the substantia nigra, interpeduncular nucleus and areas dorsolateral to the latter nucleus. Slightly higher 4
A
B
C
a
b
C
d
e
Fig. 1Aa--e. DA areas in mouse brain labeled by autoradiographic localization of 14C.NE uptake in the presence of DMI in vitro. Light areas represent radioactivity. The brain slabs are taken from approximately the telencephaIon near the anterior commissure (a), the anterior hypothalamus (b), the medial hypothalamus (c), the mesencephalon (d) and the metencephalon (e). Fig. 1Ba--e. CA areas (including NE and DA) in mouse brain labeled by autoradiographie localization of 14C-NE uptake in vitro. Fig. 1Ca--e. 5-HT areas in mouse brain labeled by autoradiographic localization of 14C-5-HT uptake in vitro. Key: 1, cerebral cortex; 2, nucleus caudatus/putamen; 3, septum; 4, hippocampus; 5, nucleus ventralis thalami; 6, globus pallidus; 7, median eminence; 8, nucleus ventromedialis hypothalami; 9, nucleus amygdaloideus; 10, cerebellum.
AUTORADIOGRAPHIC MONOAMINE LOCALIZATION
levels w e r e also o b s e r v e d in t h e central gray and
raphe. T h e cerebellar c o r t e x s h o w e d a very l o w level o f ~ 4 C-5-HT u p t a k e .
4. D i s c u s s i o n I n c u b a t i o n of coronal brain slabs with ~4 CNE resulted in an ubiquitous labeling of all CA neurons (fig. 1B) including NE, DA and possibly the newly described adrenaline neurons (H6kfelt et al., 1974b). The fact that 14 C-NE is t a k e n up into brain areas to a level roughly paralleling the density of fluorescent nerve terminals and tracts as demonstrated by the Falck--Hillarp technique (Dahlstr6m and Fuxe, 1964; Fuxe, 1965; Ungerstedt, 1971), that uptake is lacking after intraventricular 6-hydroxydopamine pretreatment (Masuoka and Alcaraz, 1973} and is inhibited after desipramine indicates that specific uptake into catecholaminergic neurons was involved. The diagonal line of terminals running ventrolaterally in the nucleus lateralis septi was identified previously as being catecholaminergic by autoradiography (Masuoka and Alcaraz, 1973) and by histofluorescence (Jacobowitz and Palkovits, 1974). With the use of DMI to block the uptake of CA into NE terminals b u t not DA terminals, this particular pattern of distribution appears in the autoradiogram to be dopaminergic. Since in a recent histochemical fluorescence study utilizing a combination of drug pretreatments (Lidbrink et al., 1974), the authors concluded that 'some of the CA terminals in the septal area may be DA nerve terminals', the present autoradiographic result provides further support for the view that these terminals indeed are dopaminergic. Other areas such as the corpus striatum observed previously in the fluorescence microscope as DA areas in the rat have been accurately labeled. The recently described DA terminals in the cerebral cortex (Thierry et al., 1973; H6kfelt et al., 1974a) were not readily apparent in these mouse slabs. At this point, it appears that the fine DA terminals in the cortex are not distin-
129
guishable with certainty by this method. The high 14 C-NE activity around the periphery or superficial layers of the cerebral cortex (Ba-e) has been discussed in detail previously (Masuoka and Alcaraz, 1973). It was concluded that high radioactivity was due to uptake into CA nerve terminals possibly in the pia or outer cortical layers (Falck et al., 1965; Fuxe et al., 1968a). The possibility of an artifact is excluded since brain slabs incubated in the absence of radioactivity and otherwise processed identically produced no traces on the X-ray film. Furthermore, after DMI pretreatment this ring of high activity, although not completely absent, was markedly reduced (Aa-e) strongly suggesting the presence of NE nerve terminals. The high 14 C-5-HT activity around the periphery of the cerebral cortex (Ca
