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A light and electron microscopic study of noradrenergic terminals in the rat dentate gyrus
Brain Research, 120 (1977) 327-335
327
© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Short Communications
A light and electron microscopic study of noradrenergic terminals in the rat dentate gyrus
LEONARD Y. KODA and FLOYD E. BLOOM Arthur Vining Davis Center for Behavioral Neurobiology, The Salk Institute, La Jolla, Calif. 92037 (U.S.A.)
(Accepted October 14th, 1976)
Small granular vesicles (SGV) associated with the biogenic amines, norepinephrine (NE), dopamine and serotonin were first demonstrated in the central nervous system by H/3kfelt8,9. These permanganate-fixed SGV-containing boutons (SGV boutons) are assumed to be the ultrastructural correlate of aldehyde-induced monoamine fluorescent varicosities as seen with the light microscope1,4,5, 9. Dopamine and serotonin associated SGVs can only be seen after intraventricular injection or in vitro incubation with exogenous amines prior to fixation 1,9. NE-associated SGVs, on the other hand, are revealed without prior exposure to exogenous amines by directly fixing brain slices in potassium permanganatel,lL Because exogenous amines are needed to demonstrate dopamine and serotonin associated SGVs, quantification of SGVs associated with these two transmitters before and after experimental treatment is difficult since their numbers might vary with the amount of exogenous amine needed to 'produce' them rather than with the experimental treatment. The ambiguities of exogenous amine incubation need not be a problem with the quantification of NEassociated SGVs (NE SGVs) but other factors do cause problems. The efficiency with which permanganate fixation reveals NE SGVs is not known; this problem could be dealt with in experimental studies by assuming the same relative efficiency in experimental and control groups. A more serious problem, possibly related to the efficiency with which permanganate reveals NE SGVs, is the infrequency with which NE SGVs are observed in the brain regions of normal animals examined thus far. Only one quantitative study of NE SGVs has been reported and it examined mutant mice which have abnormally rich NE innervation to their cerebellumlL In the present study, and a preliminary abstract 1~, the topological distribution and quantification of NE SGVs and of glyoxylic acid-induced monoamine fluorescent (GIF) varicosities is reported. We have combined the potassium permanganate fixation of Richardson 16 with the G I F method of Bloom and Battenberg 7. This combination of techniques yields reproducible numbers of SGV boutons per unit
328 area of thin section; and in addition, tissue from the same animal can be conveniently prepared for G I F to permit correlative light microscopy of NE fibers and boutons. Male Sprague-Dawley rats (150-250 g) were perfused with 250 ml of an ice-cold 2 ~ glyoxylic acid-0.5~o paraformaldehyde solution 7 in approximately 90 sec. The brain was hemisected and the dentate gyrus sliced into 1 m m sections with a chilled razor blade. Sections were immersed 30-60 min in a saturated solution of potassium permanganate (approximately 4.5 ~ at 4 °C) in Ringer's solution, rinsed with Ringer's solution, block stained in 1 ~ uranyl acetate in Ringer's solution (1-2 h at room temperature) and then processed for electron microscopy. In some experiments the contralateral hemisphere was frozen and processed for G I F by the method of Bloom and Battenberg 7. Frozen sections (20/~m) were examined for G I F varicosities with a Zeiss microscope (epi-fluorescence condenser IV; filter pack number 487705) or Nikon fluorescence microscope. Green varicose fibers were identified as NE-containing on the basis of pharmacological sensitivity 7 and correlation with previous light microscopic methods 3, 13-15,19. For quantification of G I F varicosities, 200 mesh grids (one grid square is approximately 6500 sq./zm) were placed on the G1F sections and the dentate gyrus was photographed (Kodak Tri-X) at 50 x . Enlargements (6-7 x ) were made and the varicosities within the grid squares or within masks of appropriate area were counted. Estimates of the diameter of the G | F varicosities were also made on these enlargements. Thin sections were mounted on 200 mesh grids and examined systematically at a primary magnification of × 5500 on the microscope screen. Adjacent thick sections were stained with toluidine blue and used to orient the thin sections. All boutons and SGV boutons were counted on the microscope screen. Boutons were defined as membrane bound profiles containing 5 or more synaptic vesicles. The relative cytological location of each SGV bouton was noted and then the bouton was photographed at a primary magnification of x 10,000. Bouton and vesicle analyses were made on enlargements (total magnification of x 67,000 or x 88,000) calibrated with a replica grating. The diameter of each SGV bouton was calculated as the mean of the widest and narrowest profile. In the present study, a SGV bouton is said to be in a 'synaptic cluster' if it is juxtaposed to a dendrite that is in close contact with at least one other bouton in the plane of the section (see Fig. 2a). We employed the term 'synaptic cluster' as an alternative structural property to the synaptic specializations since the latter paramembranous details are generally quite subdued after permanganate fixation. The presence of'pre-synaptic' aggregation of vesicles was also noted. Reserpine (10 mg/kg i.p., Sandril) was administered 18-24 h before perfusion. intracisternal 6-hydroxydopamine ( 6 - O H D A - H B r salt, 250 #g, Sigma) was administered under halothane anesthesis. 6 - O H D A was dissolved in 50/~1 of Ringer's solution (containing 1 mg/ml ascorbic acid). 6 - O H D A treated rats received either one dose and were perfused 18-24 h later or received two doses 24 h apart and then were perfused 3 or 7 days later. In some experiments contralateral hemispheres were prepared for either electron microscopy or G1F. For G I F, control and treated brains were processed together on the same microscope slide.
329
!
b
m
M Fig. 1. Correlation between GIF varicosities and SGV boutons in the rat dentate gyrus, a: GIF micrograph of dentate gyrus. Varicose fibers densely innervate the hilus (H) as compared to the granule cell (G) and molecular (M) layers, b: diagrammatic representation of a thin section of the dentate gyrus on a 200 mesh grid. Each dot represents a SGV bouton. Bar, 100/~m.
330 TABLE I Distribution and density o f G1F varicosities in the rat dentate gyrus
GIF varicosities (approximately 1/~m in diameter) were counted on photomicrographs masked with 6500 sq.#m grid squares.
Number of grid squares examined Varicosities(± S.E.M.)pergridsquare
Molecular layer
Granule cell layer Hilus
57 21.54 ± 1.49
29 30.66 ± 2.38*
57 85.93 zL 3.09**
* P < 0.01 with respect to molecular layer. ** P < 0.001 with respect to molecular or granule cell layers. G r e e n varicose fibers (white in Fig. 1a) occur m o r e in the hilus t h a n in the molecular or granule cell layers. The n u m b e r o f G I F varicosities p e r representative grid square is q u a n t i t a t e d in T a b l e I. The d i a m e t e r o f a varicosity is a p p r o x i m a t e l y 1 / , m . The density o f G I F varicosities p e r grid square in the hilus is significantly greater t h a n in either the m o l e c u l a r o r granule cell layers. The d i s t r i b u t i o n a n d density o f S G V b o u t o n s (as d e t e r m i n e d by electron microscopy) in the d e n t a t e gyrus is closely c o r r e l a t e d with the d i s t r i b u t i o n a n d density o f G I F varicosities (as d e t e r m i n e d by light microscopy). Fig. l b d i a g r a m m a t i c a l l y represents a thin section m o u n t e d on a 200 mesh grid. S G V b o u t o n s (dots) were f o u n d p r e d o m i n a n t l y within the d e n t a t e hilus. D a t a f r o m three rats are presented in T a b l e I1. The density o f S G V b o u t o n s is expressed as a function o f a r e a ( S G V b o u t o n s per grid square) a n d as a function o f total b o u t o n s o b s e r v e d ( % S G V boutons). Both measures o f density yield significantly greater densities o f S G V b o u t o n s in the hilus as c o m p a r e d to either the m o l e c u l a r o r granule cell layers. The relative n u m b e r s o f S G V b o u t o n s p e r unit a r e a are in line with n u m b e r s expected by e x t r a p o l a t i o n f r o m G I F : a p p r o x i m ately 85 G I F varicosities occur p e r 6500 sq./~m in a 20 # m frozen section (Table I); a s s u m i n g a 1 # m d i a m e t e r varicosity (Table I), 4 S G V b o u t o n s w o u l d be expected TABLE II Distribution and density o f small granular vesicle containing boutons in the rat dentate gyrus
Data from three control rats are presented in this table. Sections were mounted on 200 mesh grids (approximate area of one grid square is 6500 sq./zm) and one section from each animal was examined.
Number of grid squares examined Total SGV boutons observed Total boutons observed SGVboutons(± S.E.M.)pergridsquare Per cent of boutons (4- S.E.M.) that are SGV boutons
Molecular layer
Granule cell layer Hilus
58 27 134,984 0.47 ± 0.09
15 14 8,667 0.93 -I- 0.32
23 71 33,857 3.09 ± 0.39*
0.02 ± 0.004**
0.18 ± 0.07
0.27 -L 0.05
* P < 0.001 with respect to molecular or granule cell layers. ** P < 0.05 with respect to granule cell layer or hilus.
331
TABLE Ill Diameter of SG V boutons
Ninety-three SGV boutons from three control rats are presented in this table. Bouton diameter (l~m)
%
Diameter of SG V boutons ( ± S.E.M:)
0.4-0.59 0.6-0.79 0.8-0.99 1.0-1.19 1.2-1.39 1.4-1.59
22 28 28 16 5 0
0.82 4- 0.3
1.6-1.79
1
per 6500 sq.#m in a 60 nm thin section, and 3 SGV boutons per 6500 sq./~m (Table II) were seen. The above calculation assumes that a thin section of potassium permanganate fixed tissue will reveal all 1/~m varicosities. The mean diameter (4- S.E.M.) of the SGV boutons examined in this study was 0.82 4- 0.3/~m (Table III). There were 20.57 4- 1.28 vesicles per SGV bouton in the 3 random sections examined (Table IV). Fifty-five per cent of the vesicles were SGV (approximately 50 nm in diameter), 16% were small, agranular, while 22% of the vesicles were small but not classified as either granular or agranular because of technical reasons (i.e. the plane of section or focus did not pexmit an unambiguous assessment of granularity). Large vesicles (approximately 120 nm in diameter) comprised only 6 % of the vesicles population. N o difference in the various vesicle populations in boutons containing SGVs was noted between the three layers of the dentate gyrus. As shown in Table V, approximately 20 % of the SGV boutons observed in both the molecular layer and hilus of the fascia dentata were in synaptic clusters while none seen TABLE IV Vesicles within SG V boutons
Data from three rats are presented in this table. Numbers in parentheses equal the number of observed vesicles. Vesicles (4- S.E.M.) per bouton
Small (approximately 50 nm diameter) granular 11.39 4- 0.65 agranular 3.38 4- 0.37 not classified 4.44 + 0.45 Large (approximately 125 nm diameter) granular 0.51 4- 0.09 agranular 0.24 4- 0.08 not classified 0.60 4- 0.10
(1093) (324) (426) (49) (23) (58)
%
55 16 22 2 1 3
332 TABLE V Distribution o f synaptie clusters in the rat dentate gyrus
Data from three control rats are presented in this table.
Number of SGV boutons observed Per cent ofSGV boutons in synaptic clusters
Molecular layer
Granule cell layer Hilus
27
12
57
19
0
18
in the granule cell layer were in such clusters. One-half of the SGV boutons in synaptic clusters seen in the hilus displayed 'presynaptic' aggregation of vesicles (see Fig. 2a). Reserpine totally depleted the dentate hilus of SGV boutons at 18 h (Table VI). Two intracisternal injections of 6 - O H D A significantly decreased the number of SGV boutons per unit area in the dentate hilus after 3 or 7 days (Table VI). One day after one dose of 6 - O H D A normal numbers of SGV boutons were observed but early degenerative changes may have begun in these SGV boutons (see Fig. 2c). No G I F varicosities were observed in the dentate gyrus one day after reserpine or 6 - O H D A treatment; instead a diffuse but reduced fluorescence was seen. In the present study, the topological distribution and quantitation of SGV boutons in the normal rat dentate gyrus is reported. The distribution, density and relative size of the SGV boutons correlate closely with the distribution, density and relative size of the G I F varicosities. Intraperitoneal injections of reserpine or intracisternal injections of 6 - O H D A deplete the dentate hilus of SGV boutons and G I F varicosities. Depletion of SGV boutons by reserpine a,u combined with depletion by 6 - O H D A (Table VI) is strong evidence that SGV boutons are central NE-containing fibers and terminals 5,9,1°. The fact that SGV boutons were not depleted 24 h after 6 - O H D A (a time at which G I F was diffuse) may be explained by several factors. (1) 6-OHDA, like 5-hydroxydopamine, is taken up by nerve terminals and can produce granulated vesicles 2,17. (2) Degenerative effects of 6 - O H D A (see Fig. 2c) may have just TABLE VI Effect o f various drug treatments on the number o f SG V boutons per unit area in the hilus o f the rat dentate gyrus
Five grid squares (approximately 6500 sq./~m in area) near the apex of the hilus were examined from each animal.
SGV boutons (4- S.E.M.) of per grid square Number of grid squares examined * P < 0,001.
Control
Reserpine
1 day 6-OHDA
3 day 6-OHDA
7 day 6-OHDA
3.8 i 0.47
0.0 ± 0.0"
3.4 ± 0.58
0.3 i 0.15" 0.9 ± 0.31"
15
15
15
l0
10
333
Fig. 2. Electron micrographs of SGV boutons in the rat dentate gyrus, a: SGV bouton in a synaptic cluster. The arrow is in a dendrite and points towards a SGV bouton with 'presynaptic' aggregation of vesicles, b: two SGV boutons 24 h after 6-OHDA. The small bouton appears normal while the larger one contains membrane fragments, c: two apparently normal SGV boutons 24 h after 6-OHDA. Arrows indicate avesicular varicosities and fibers that connect two SGV boutons. The lower SGV bouton is in a synaptic cluster. Bar, 1/~m. b e g u n to manifest themselves at 24 h in this particular region, somewhat r e m o v e d from the ventricular system is, or (3) electron microscopy m a y well be more sensitive t h a n G I F in detecting central N E b o u t o n s u n d e r these conditions. M o s t of the S G V b o u t o n s e x a m i n e d in the present study are p r o b a b l y fibers in passage while some m a y be synapses. Because p o t a s s i u m p e r m a n g a n a t e does n o t stain
334
synaptic clefts prominently in our preparations, other criteria were used to identify possible synapses (see methods for definition of synaptic cluster). SGV boutons in synaptic clusters were not seen in the granule cell layer. Since 20 ~ of the SGV boutons in the molecular layer and hilus were in synaptic clusters, SGV boutons probably synapse there. The presence of a SGV bouton in a synaptic cluster does not positively identify a synapse; even when combined with 'presynaptic aggregation' of vesicles it is only suggestive of a synapse. On the other hand, the lack o f a synaptic cluster does not rule out the presence of a synapse since only serial sectioning can reveal all synaptic clusters. It should be kept in mind however, that as is seen in the peripheral nervous system 4, release of norepinephrine might well occur without synaptic profiles. We have combined GIF and KMnO4 fixation into a correlative light and electron microscopic study of the NE terminals in the rat dentate gyrus. The hippocampus was chosen for this study because of its rich NE innervation from the locus coeruleus 3,1a-15, 19 and because of its highly organized structure. Further studies are in progress to demonstrate the usefulness of the combined techniques and of the hippocampus as an ultrastructural model to study central NE mechanisms. Supported in part by grants from the Alfred P. Sloan Foundation and by the National Science Foundation (BNS-76-09318).
1 Ajika, K. and Hbkfelt, T., Ultrastructural identification of catecholamine neurones in the hypothalamic periventricular-arcuate nucleus-median eminence complex with special reference to quantitative aspects, Brain Research, 57 (1973) 97-117. 2 Bennett, T., Burnstock, G., Cobb, J. L. S. and Malmfors, T., An ultrastructural and histochemical study of the short-term effects of 6-hydroxydopamine on adrenergic nerves in domestic fowl, Brit. J. Pharmacol., 38 (1970) 802-809. 3 Blackstad, T. W., Fuxe, K. and Hbkfelt, T., Noradrenaline nerve terminals in the hippocampal region of the rat and guinea pig, Z. Zellforsch., 78 (1967) 463-473. 4 Bloom, F. E., Electron microscopy of catecholamine-containing structures. In H. Blaschko and E. MuschoU (Eds.), Handbook of Experimental Pharmacology, Vol. XXXIII, Springer, Berlin, 1972, pp. 46-78. 5 Bloom, F. E., Ultrastructural identification ofcatecholamine-containing central synaptic terminals, J. Histochem. Cytochem., 21 (1973) 333-348. 6 Bloom, F. E., Algeri, S., Gropetti, A., Revuelta, A. and Costa, E., Science, 166 (1969) 1284-1286. 7 Bloom, F. E. and Battenberg, E. L. F., A rapid, simple and sensitive method for the demonstration of central catecholamine-containing neurons and axons by glyoxylic acid-induced fluorescence, J. Histochem. Cytochem., 24 (1976) 561-571. 8 Hbkfelt, T., On the ultrastructural localization of noradrenaline in the central nervous system of the rat, Z. Zellforsch., 79 (1967) 110-117. 9 H6kfelt, T., In vitro studies on central and peripheral monoamine neurons at the ultrastructural level, Z. Zellforsch., 91 (1968) 1-74. 10 Jacks, B. R., De Champlain, J. and Cordeau, J. P., Effects of 6-hydroxydopamine on putative transmitter substances in the central nervous system, Europ. J. Pharmacol., 18 (1972) 353-360. 11 Koda, L. Y. and Bloom, F. E., Small granular vesicles in the rat dentate gyrus, Anat. Rec., 184 (1976) 450~51. 12 Landis, S. C., and Bloom, F. E., U ttrastructural identification of noradrenergic boutons in mutant and normal mouse cerebellar cortex, Brain Research, 96 (1975) 299-305. 13 Loizou, L. A., Projections of the nucleus locus coeruleus in the albino rat, Brain, 15 (1969) 563-566. 14 Moore, R. Y., Monoamine neurons innervating the hippocampal formation and the septum: organization and response to injury. In R. L. lsaacson and K. H. Pribram (Eds.), The Hippocampus, FoL 1, Plenum Press, New York, 1975, pp. 215-237.
335 15 Pickel, V. M., Segal, M. and Bloom, F. E., An radioautographic study of the efferent pathways of the nucleus locus coeruleus, J. comp. Neurol., 155 (1974) 15--42. 16 Richardson, K. C., Electron microscopic identification of autonomic nerve endings, Nature (Lond.), 210 (1966) 756. 17 Tranzer, J. P. and Richards, J. G., Fine structural aspects of 6-hydroxydopamine of peripheral adrenergic neurons. In T. Malmfors and H. Thoenen (Eds.), 6-Hydroxydopamine, North Holland Publ., Amsterdam, 1971, pp. 15-31. 18 Ungerstedt, U., Histochemical studies of the effect of intracerebral and intraventricular injections of 6-hydroxydopamine on monoamine neurons in the rat brain. In T. Malmfors and H. Thoenen (Eds.), 6-Hydroxydopamine, North Holland Publ., Amsterdam, 1971, pp. 101-127. 19 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physioL scand., 367, Suppl. (1971) 1-48.
327
© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Short Communications
A light and electron microscopic study of noradrenergic terminals in the rat dentate gyrus
LEONARD Y. KODA and FLOYD E. BLOOM Arthur Vining Davis Center for Behavioral Neurobiology, The Salk Institute, La Jolla, Calif. 92037 (U.S.A.)
(Accepted October 14th, 1976)
Small granular vesicles (SGV) associated with the biogenic amines, norepinephrine (NE), dopamine and serotonin were first demonstrated in the central nervous system by H/3kfelt8,9. These permanganate-fixed SGV-containing boutons (SGV boutons) are assumed to be the ultrastructural correlate of aldehyde-induced monoamine fluorescent varicosities as seen with the light microscope1,4,5, 9. Dopamine and serotonin associated SGVs can only be seen after intraventricular injection or in vitro incubation with exogenous amines prior to fixation 1,9. NE-associated SGVs, on the other hand, are revealed without prior exposure to exogenous amines by directly fixing brain slices in potassium permanganatel,lL Because exogenous amines are needed to demonstrate dopamine and serotonin associated SGVs, quantification of SGVs associated with these two transmitters before and after experimental treatment is difficult since their numbers might vary with the amount of exogenous amine needed to 'produce' them rather than with the experimental treatment. The ambiguities of exogenous amine incubation need not be a problem with the quantification of NEassociated SGVs (NE SGVs) but other factors do cause problems. The efficiency with which permanganate fixation reveals NE SGVs is not known; this problem could be dealt with in experimental studies by assuming the same relative efficiency in experimental and control groups. A more serious problem, possibly related to the efficiency with which permanganate reveals NE SGVs, is the infrequency with which NE SGVs are observed in the brain regions of normal animals examined thus far. Only one quantitative study of NE SGVs has been reported and it examined mutant mice which have abnormally rich NE innervation to their cerebellumlL In the present study, and a preliminary abstract 1~, the topological distribution and quantification of NE SGVs and of glyoxylic acid-induced monoamine fluorescent (GIF) varicosities is reported. We have combined the potassium permanganate fixation of Richardson 16 with the G I F method of Bloom and Battenberg 7. This combination of techniques yields reproducible numbers of SGV boutons per unit
328 area of thin section; and in addition, tissue from the same animal can be conveniently prepared for G I F to permit correlative light microscopy of NE fibers and boutons. Male Sprague-Dawley rats (150-250 g) were perfused with 250 ml of an ice-cold 2 ~ glyoxylic acid-0.5~o paraformaldehyde solution 7 in approximately 90 sec. The brain was hemisected and the dentate gyrus sliced into 1 m m sections with a chilled razor blade. Sections were immersed 30-60 min in a saturated solution of potassium permanganate (approximately 4.5 ~ at 4 °C) in Ringer's solution, rinsed with Ringer's solution, block stained in 1 ~ uranyl acetate in Ringer's solution (1-2 h at room temperature) and then processed for electron microscopy. In some experiments the contralateral hemisphere was frozen and processed for G I F by the method of Bloom and Battenberg 7. Frozen sections (20/~m) were examined for G I F varicosities with a Zeiss microscope (epi-fluorescence condenser IV; filter pack number 487705) or Nikon fluorescence microscope. Green varicose fibers were identified as NE-containing on the basis of pharmacological sensitivity 7 and correlation with previous light microscopic methods 3, 13-15,19. For quantification of G I F varicosities, 200 mesh grids (one grid square is approximately 6500 sq./zm) were placed on the G1F sections and the dentate gyrus was photographed (Kodak Tri-X) at 50 x . Enlargements (6-7 x ) were made and the varicosities within the grid squares or within masks of appropriate area were counted. Estimates of the diameter of the G | F varicosities were also made on these enlargements. Thin sections were mounted on 200 mesh grids and examined systematically at a primary magnification of × 5500 on the microscope screen. Adjacent thick sections were stained with toluidine blue and used to orient the thin sections. All boutons and SGV boutons were counted on the microscope screen. Boutons were defined as membrane bound profiles containing 5 or more synaptic vesicles. The relative cytological location of each SGV bouton was noted and then the bouton was photographed at a primary magnification of x 10,000. Bouton and vesicle analyses were made on enlargements (total magnification of x 67,000 or x 88,000) calibrated with a replica grating. The diameter of each SGV bouton was calculated as the mean of the widest and narrowest profile. In the present study, a SGV bouton is said to be in a 'synaptic cluster' if it is juxtaposed to a dendrite that is in close contact with at least one other bouton in the plane of the section (see Fig. 2a). We employed the term 'synaptic cluster' as an alternative structural property to the synaptic specializations since the latter paramembranous details are generally quite subdued after permanganate fixation. The presence of'pre-synaptic' aggregation of vesicles was also noted. Reserpine (10 mg/kg i.p., Sandril) was administered 18-24 h before perfusion. intracisternal 6-hydroxydopamine ( 6 - O H D A - H B r salt, 250 #g, Sigma) was administered under halothane anesthesis. 6 - O H D A was dissolved in 50/~1 of Ringer's solution (containing 1 mg/ml ascorbic acid). 6 - O H D A treated rats received either one dose and were perfused 18-24 h later or received two doses 24 h apart and then were perfused 3 or 7 days later. In some experiments contralateral hemispheres were prepared for either electron microscopy or G1F. For G I F, control and treated brains were processed together on the same microscope slide.
329
!
b
m
M Fig. 1. Correlation between GIF varicosities and SGV boutons in the rat dentate gyrus, a: GIF micrograph of dentate gyrus. Varicose fibers densely innervate the hilus (H) as compared to the granule cell (G) and molecular (M) layers, b: diagrammatic representation of a thin section of the dentate gyrus on a 200 mesh grid. Each dot represents a SGV bouton. Bar, 100/~m.
330 TABLE I Distribution and density o f G1F varicosities in the rat dentate gyrus
GIF varicosities (approximately 1/~m in diameter) were counted on photomicrographs masked with 6500 sq.#m grid squares.
Number of grid squares examined Varicosities(± S.E.M.)pergridsquare
Molecular layer
Granule cell layer Hilus
57 21.54 ± 1.49
29 30.66 ± 2.38*
57 85.93 zL 3.09**
* P < 0.01 with respect to molecular layer. ** P < 0.001 with respect to molecular or granule cell layers. G r e e n varicose fibers (white in Fig. 1a) occur m o r e in the hilus t h a n in the molecular or granule cell layers. The n u m b e r o f G I F varicosities p e r representative grid square is q u a n t i t a t e d in T a b l e I. The d i a m e t e r o f a varicosity is a p p r o x i m a t e l y 1 / , m . The density o f G I F varicosities p e r grid square in the hilus is significantly greater t h a n in either the m o l e c u l a r o r granule cell layers. The d i s t r i b u t i o n a n d density o f S G V b o u t o n s (as d e t e r m i n e d by electron microscopy) in the d e n t a t e gyrus is closely c o r r e l a t e d with the d i s t r i b u t i o n a n d density o f G I F varicosities (as d e t e r m i n e d by light microscopy). Fig. l b d i a g r a m m a t i c a l l y represents a thin section m o u n t e d on a 200 mesh grid. S G V b o u t o n s (dots) were f o u n d p r e d o m i n a n t l y within the d e n t a t e hilus. D a t a f r o m three rats are presented in T a b l e I1. The density o f S G V b o u t o n s is expressed as a function o f a r e a ( S G V b o u t o n s per grid square) a n d as a function o f total b o u t o n s o b s e r v e d ( % S G V boutons). Both measures o f density yield significantly greater densities o f S G V b o u t o n s in the hilus as c o m p a r e d to either the m o l e c u l a r o r granule cell layers. The relative n u m b e r s o f S G V b o u t o n s p e r unit a r e a are in line with n u m b e r s expected by e x t r a p o l a t i o n f r o m G I F : a p p r o x i m ately 85 G I F varicosities occur p e r 6500 sq./~m in a 20 # m frozen section (Table I); a s s u m i n g a 1 # m d i a m e t e r varicosity (Table I), 4 S G V b o u t o n s w o u l d be expected TABLE II Distribution and density o f small granular vesicle containing boutons in the rat dentate gyrus
Data from three control rats are presented in this table. Sections were mounted on 200 mesh grids (approximate area of one grid square is 6500 sq./zm) and one section from each animal was examined.
Number of grid squares examined Total SGV boutons observed Total boutons observed SGVboutons(± S.E.M.)pergridsquare Per cent of boutons (4- S.E.M.) that are SGV boutons
Molecular layer
Granule cell layer Hilus
58 27 134,984 0.47 ± 0.09
15 14 8,667 0.93 -I- 0.32
23 71 33,857 3.09 ± 0.39*
0.02 ± 0.004**
0.18 ± 0.07
0.27 -L 0.05
* P < 0.001 with respect to molecular or granule cell layers. ** P < 0.05 with respect to granule cell layer or hilus.
331
TABLE Ill Diameter of SG V boutons
Ninety-three SGV boutons from three control rats are presented in this table. Bouton diameter (l~m)
%
Diameter of SG V boutons ( ± S.E.M:)
0.4-0.59 0.6-0.79 0.8-0.99 1.0-1.19 1.2-1.39 1.4-1.59
22 28 28 16 5 0
0.82 4- 0.3
1.6-1.79
1
per 6500 sq.#m in a 60 nm thin section, and 3 SGV boutons per 6500 sq./~m (Table II) were seen. The above calculation assumes that a thin section of potassium permanganate fixed tissue will reveal all 1/~m varicosities. The mean diameter (4- S.E.M.) of the SGV boutons examined in this study was 0.82 4- 0.3/~m (Table III). There were 20.57 4- 1.28 vesicles per SGV bouton in the 3 random sections examined (Table IV). Fifty-five per cent of the vesicles were SGV (approximately 50 nm in diameter), 16% were small, agranular, while 22% of the vesicles were small but not classified as either granular or agranular because of technical reasons (i.e. the plane of section or focus did not pexmit an unambiguous assessment of granularity). Large vesicles (approximately 120 nm in diameter) comprised only 6 % of the vesicles population. N o difference in the various vesicle populations in boutons containing SGVs was noted between the three layers of the dentate gyrus. As shown in Table V, approximately 20 % of the SGV boutons observed in both the molecular layer and hilus of the fascia dentata were in synaptic clusters while none seen TABLE IV Vesicles within SG V boutons
Data from three rats are presented in this table. Numbers in parentheses equal the number of observed vesicles. Vesicles (4- S.E.M.) per bouton
Small (approximately 50 nm diameter) granular 11.39 4- 0.65 agranular 3.38 4- 0.37 not classified 4.44 + 0.45 Large (approximately 125 nm diameter) granular 0.51 4- 0.09 agranular 0.24 4- 0.08 not classified 0.60 4- 0.10
(1093) (324) (426) (49) (23) (58)
%
55 16 22 2 1 3
332 TABLE V Distribution o f synaptie clusters in the rat dentate gyrus
Data from three control rats are presented in this table.
Number of SGV boutons observed Per cent ofSGV boutons in synaptic clusters
Molecular layer
Granule cell layer Hilus
27
12
57
19
0
18
in the granule cell layer were in such clusters. One-half of the SGV boutons in synaptic clusters seen in the hilus displayed 'presynaptic' aggregation of vesicles (see Fig. 2a). Reserpine totally depleted the dentate hilus of SGV boutons at 18 h (Table VI). Two intracisternal injections of 6 - O H D A significantly decreased the number of SGV boutons per unit area in the dentate hilus after 3 or 7 days (Table VI). One day after one dose of 6 - O H D A normal numbers of SGV boutons were observed but early degenerative changes may have begun in these SGV boutons (see Fig. 2c). No G I F varicosities were observed in the dentate gyrus one day after reserpine or 6 - O H D A treatment; instead a diffuse but reduced fluorescence was seen. In the present study, the topological distribution and quantitation of SGV boutons in the normal rat dentate gyrus is reported. The distribution, density and relative size of the SGV boutons correlate closely with the distribution, density and relative size of the G I F varicosities. Intraperitoneal injections of reserpine or intracisternal injections of 6 - O H D A deplete the dentate hilus of SGV boutons and G I F varicosities. Depletion of SGV boutons by reserpine a,u combined with depletion by 6 - O H D A (Table VI) is strong evidence that SGV boutons are central NE-containing fibers and terminals 5,9,1°. The fact that SGV boutons were not depleted 24 h after 6 - O H D A (a time at which G I F was diffuse) may be explained by several factors. (1) 6-OHDA, like 5-hydroxydopamine, is taken up by nerve terminals and can produce granulated vesicles 2,17. (2) Degenerative effects of 6 - O H D A (see Fig. 2c) may have just TABLE VI Effect o f various drug treatments on the number o f SG V boutons per unit area in the hilus o f the rat dentate gyrus
Five grid squares (approximately 6500 sq./~m in area) near the apex of the hilus were examined from each animal.
SGV boutons (4- S.E.M.) of per grid square Number of grid squares examined * P < 0,001.
Control
Reserpine
1 day 6-OHDA
3 day 6-OHDA
7 day 6-OHDA
3.8 i 0.47
0.0 ± 0.0"
3.4 ± 0.58
0.3 i 0.15" 0.9 ± 0.31"
15
15
15
l0
10
333
Fig. 2. Electron micrographs of SGV boutons in the rat dentate gyrus, a: SGV bouton in a synaptic cluster. The arrow is in a dendrite and points towards a SGV bouton with 'presynaptic' aggregation of vesicles, b: two SGV boutons 24 h after 6-OHDA. The small bouton appears normal while the larger one contains membrane fragments, c: two apparently normal SGV boutons 24 h after 6-OHDA. Arrows indicate avesicular varicosities and fibers that connect two SGV boutons. The lower SGV bouton is in a synaptic cluster. Bar, 1/~m. b e g u n to manifest themselves at 24 h in this particular region, somewhat r e m o v e d from the ventricular system is, or (3) electron microscopy m a y well be more sensitive t h a n G I F in detecting central N E b o u t o n s u n d e r these conditions. M o s t of the S G V b o u t o n s e x a m i n e d in the present study are p r o b a b l y fibers in passage while some m a y be synapses. Because p o t a s s i u m p e r m a n g a n a t e does n o t stain
334
synaptic clefts prominently in our preparations, other criteria were used to identify possible synapses (see methods for definition of synaptic cluster). SGV boutons in synaptic clusters were not seen in the granule cell layer. Since 20 ~ of the SGV boutons in the molecular layer and hilus were in synaptic clusters, SGV boutons probably synapse there. The presence of a SGV bouton in a synaptic cluster does not positively identify a synapse; even when combined with 'presynaptic aggregation' of vesicles it is only suggestive of a synapse. On the other hand, the lack o f a synaptic cluster does not rule out the presence of a synapse since only serial sectioning can reveal all synaptic clusters. It should be kept in mind however, that as is seen in the peripheral nervous system 4, release of norepinephrine might well occur without synaptic profiles. We have combined GIF and KMnO4 fixation into a correlative light and electron microscopic study of the NE terminals in the rat dentate gyrus. The hippocampus was chosen for this study because of its rich NE innervation from the locus coeruleus 3,1a-15, 19 and because of its highly organized structure. Further studies are in progress to demonstrate the usefulness of the combined techniques and of the hippocampus as an ultrastructural model to study central NE mechanisms. Supported in part by grants from the Alfred P. Sloan Foundation and by the National Science Foundation (BNS-76-09318).
1 Ajika, K. and Hbkfelt, T., Ultrastructural identification of catecholamine neurones in the hypothalamic periventricular-arcuate nucleus-median eminence complex with special reference to quantitative aspects, Brain Research, 57 (1973) 97-117. 2 Bennett, T., Burnstock, G., Cobb, J. L. S. and Malmfors, T., An ultrastructural and histochemical study of the short-term effects of 6-hydroxydopamine on adrenergic nerves in domestic fowl, Brit. J. Pharmacol., 38 (1970) 802-809. 3 Blackstad, T. W., Fuxe, K. and Hbkfelt, T., Noradrenaline nerve terminals in the hippocampal region of the rat and guinea pig, Z. Zellforsch., 78 (1967) 463-473. 4 Bloom, F. E., Electron microscopy of catecholamine-containing structures. In H. Blaschko and E. MuschoU (Eds.), Handbook of Experimental Pharmacology, Vol. XXXIII, Springer, Berlin, 1972, pp. 46-78. 5 Bloom, F. E., Ultrastructural identification ofcatecholamine-containing central synaptic terminals, J. Histochem. Cytochem., 21 (1973) 333-348. 6 Bloom, F. E., Algeri, S., Gropetti, A., Revuelta, A. and Costa, E., Science, 166 (1969) 1284-1286. 7 Bloom, F. E. and Battenberg, E. L. F., A rapid, simple and sensitive method for the demonstration of central catecholamine-containing neurons and axons by glyoxylic acid-induced fluorescence, J. Histochem. Cytochem., 24 (1976) 561-571. 8 Hbkfelt, T., On the ultrastructural localization of noradrenaline in the central nervous system of the rat, Z. Zellforsch., 79 (1967) 110-117. 9 H6kfelt, T., In vitro studies on central and peripheral monoamine neurons at the ultrastructural level, Z. Zellforsch., 91 (1968) 1-74. 10 Jacks, B. R., De Champlain, J. and Cordeau, J. P., Effects of 6-hydroxydopamine on putative transmitter substances in the central nervous system, Europ. J. Pharmacol., 18 (1972) 353-360. 11 Koda, L. Y. and Bloom, F. E., Small granular vesicles in the rat dentate gyrus, Anat. Rec., 184 (1976) 450~51. 12 Landis, S. C., and Bloom, F. E., U ttrastructural identification of noradrenergic boutons in mutant and normal mouse cerebellar cortex, Brain Research, 96 (1975) 299-305. 13 Loizou, L. A., Projections of the nucleus locus coeruleus in the albino rat, Brain, 15 (1969) 563-566. 14 Moore, R. Y., Monoamine neurons innervating the hippocampal formation and the septum: organization and response to injury. In R. L. lsaacson and K. H. Pribram (Eds.), The Hippocampus, FoL 1, Plenum Press, New York, 1975, pp. 215-237.
335 15 Pickel, V. M., Segal, M. and Bloom, F. E., An radioautographic study of the efferent pathways of the nucleus locus coeruleus, J. comp. Neurol., 155 (1974) 15--42. 16 Richardson, K. C., Electron microscopic identification of autonomic nerve endings, Nature (Lond.), 210 (1966) 756. 17 Tranzer, J. P. and Richards, J. G., Fine structural aspects of 6-hydroxydopamine of peripheral adrenergic neurons. In T. Malmfors and H. Thoenen (Eds.), 6-Hydroxydopamine, North Holland Publ., Amsterdam, 1971, pp. 15-31. 18 Ungerstedt, U., Histochemical studies of the effect of intracerebral and intraventricular injections of 6-hydroxydopamine on monoamine neurons in the rat brain. In T. Malmfors and H. Thoenen (Eds.), 6-Hydroxydopamine, North Holland Publ., Amsterdam, 1971, pp. 101-127. 19 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physioL scand., 367, Suppl. (1971) 1-48.