Neuroscience Vol. 42, NO. 2, pp. 463472, 1991 Printed in Great Britain
0306-4522/91 $3.00 + 0.00 Pergamon Press plc © 1991 IBRO
SUBCELLULAR LOCALIZATION OF NERVE GROWTH FACTOR RECEPTORS IN IDENTIFIED CELLS OF THE RAT N U C L E U S BASALIS MAGNOCELLULARIS: A N IMMUNOCYTOCHEMICAL STUDY R. MARTINEz-MuRILLO,*'~ T. FERNANDEZ,I"M. M. ALGUACIL,t F. AGUADO,'~ M. ACHAVAL,~ P. BOVOLENTA,§J. RODRIGO'I"and M. NIETO-SAMPEDRO~ i'Unidad de Neuroanatomia y Unidad de Plasticidad Neuronal, §Instituto Cajal, C.S.I.C., Doctor Arce 37. 28002-Madrid, Spain Abstract--The subcellular location of nerve growth factor receptor in the ventromedial portion of rat globus pallidus was investigated with affinity-purified monoclonal 192-IgG following the unlabelled antibody peroxidase-antiperoxidase immunocytochemical procedure. At the light microscopic level, punctate immunoreaction product was observed in the perinuclear region and in the plasma membrane of large, probably cholinergic neurons. Examination in the electron microscope of these neurons confirmed that nerve growth factor receptor-stained cells were basal forebrain cholinergic neurons. Within these cells, immunostaining occurred in the Golgi apparatus, in multivesieular bodies and, occasionally, in rough endoplasmic reticulum cisternae and the nuclear envelope. Moreover, patches of immunoreactivity were observed associated with the outer surface of the plasma membrane of the soma and their proximal dendrites and also with the plasma membrane of distal dendrites showing scarcity of synaptic input. Positive immunostaining was never observed in synaptic clefts, but filled the space between the plasma membranes of immunoreactive neurons and those of thin glial processes in their vicinity. The location of membrane nerve growth factor receptor in close apposition to membranes of neighbouring astrocytes rather than near synaptic complexes, suggests that glial cells may be a physiological source of nerve growth factor.
s h o w n 3'7'13'18 that a large proportion of magnocellular basal forebrain neurons contains both N G F R and cholinergic enzymes. However, there is little information regarding the subcellular location of the receptor in these cells. 16 Here, we have used an afffinity-purified mouse monoclonal antibody (192IgG) to rat N G F R 5 to elucidate the electron microscopic characteristics of identified N G F R - p o s i t i v e neurons in the ventromedial portion of the globus pallidus (GP), and to visualize the subcellular location of N G F R in these cells.
Nerve growth factor ( N G F ) is a well-characterized neuronotrophic protein essential for the development, survival and maintenance of function of sympathetic- and neural crest-derived sensory neurons in the peripheral nervous system (PNS) (for reviews see Refs 11,22, 31) and of cholinergic neurons in the C N S (for review see Refs 30, 35). It also plays a critical role regulating the size of cell body, process extension and content of selected proteins, including choline acetyltransferase (CHAT), acetylcholinesterase (ACHE) and nerve growth factor receptor ( N G F R ) , of adult septal cholinergic neurons after axotomy. ~2 The presence of N G F R in basal forebrain magnocellular neurons of both rat and human has been amply documented at the light microscopic level. 1'3'6'9'13'15"17'24'28'36 Moreover, it has also been
EXPERIMENTAL PROCEDURES
Animals and tissue preparation
*To whom correspondence should be addressed. :~Present address: Departamento de Cirncias Morfol6gicas, Instituto de Biocirncias, Universidade Federal do Rio Grande do Sul. Porto Alegre, Brasil. Abbreviations: ACHE, acetylcholinesterase; CHAT, choline acetyltransferase; DAB, 3,3'-diaminobenzidine; GA, Golgi apparatus; GP, globus pallidus; NBM, nucleus basalis magnocellularis; NGF, nerve growth factor; NGFR(s), nerve growth factor receptors; NGFR-IR, nerve growth factor receptor immunoreactivity; PAP, peroxidase-antiperoxidase; PB, phosphate buffer; PBS, phosphate-buffered saline; PNS, peripheral nervous system; RER, rough endoplasmic reticulum. 463
Experiments were performed on six adult, male, albino Wistar rats (Charles River) of approximately 250 g. Under deep anaesthesia (Equithesin, Jansen Lab., 2.5 ml/kg intraperitoneally), animals were ventilated and perfused through the aorta with 0.9% saline followed by 400 ml of a fixative solution containing 4% paraformaldehyde, 0.1% glutaraldehyde and 0.03% CaC12 in 0.1 M phosphate buffer (PB) pH 7.4. Perfused brains were removed and cut into small blocks. Blocks with the selected areas were postfixed for 4h (4% paraformaldehyde in 0.1 M PB) at room temperature and cryoprotected by immersion in 30% sucrose solution in 0.1 M PB at 4°C. Nerve growth factor receptor immunocytochemistry Light microscopy. Studies were performed on two animals. After fixation and cryoprotection, serial 40-/~m-thick
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frontal frozen sections were prepared with a Leitz sledge microtome. Sections were rinsed in phosphate-buffered saline (PBS) and processed for NGFR immunocytochemistry using affinity-purified mouse monoclonal antibody (192-IgG) to rat NGFR, 5 following the peroxidaseantiperoxidase (PAP) procedure. Free-floating sections were incubated in the primary antibody (final concentration 5 #g/ml, in PBS containing 0.01% sodium azide and 0.2% Triton X-100), overnight with continuous agitation at 4°C. The remaining operations were performed at room temperature. Tissue sections were washed several times in PBS, incubated with rabbit anti-mouse IgG (Biomakor, bm; 1: 10 in PBS) for 1 h, washed again in PBS and incubated in monoclonal mouse PAP antibody (Biomakor, bm; 1: 1000 in PBS) for 90 min. The immunocytochemical reaction was revealed by incubating the tissue sections in a histochemical medium that contained 0.06% 3,Y-diaminobenzidine (DAB) tetra HC1 (Sigma, St Louis, MO) in PBS, for I0 min, and then in the same solution containing 0.003% H202. The DAB reaction was interrupted at times chosen by inspection of trial sections (approximately 8 min). Electron microscopy. Electron microscopic studies were performed on four animals. Cryoprotected blocks were rapidly frozen with liquid nitrogen and thawed in cold 0.1 M PB to improve the antibody penetration. Sections (40/~m thick) were cut with a Vibratome (Lancer) and immunostained as for light microscopy except that Triton X-100 was not included in the incubation solutions. The double bridge procedure 32 was used and incubations with rabbit antimouse IgG and mouse PAP were repeated, after washing, using the same dilutions of reagents as were used in the first bridging step (30 min each). After the DAB reaction, sections were washed in PBS (I h), postfixed in 1% osmium tetroxide in 0.1 M PB, pH 7.4 (1 h), dehydrated in ethanols and block-stained in uranyl acetate (1% in 70% ethanol) in the dark for 40 min at room temperature. The sections were mounted on Durcupan ACM resin (Fluka) slides under a plastic coverslip and cured for three days at 56°C. Selected areas of the ventromedial part of the GP, containing NGFR-positive neurons, were dissected out, re-embedded in Durcupan in plastic capsules and semithin sections (2-2.5/~m thick) were then prepared. Selected semithin sections were re-embedded in Durcupan in plastic capsules. Ultrathin serial sections were then obtained, mounted on Formvar-coated grids, stained with lead citrate and examined in a Jeol 1200 EX electron microscope. Control sections were prepared either by omitting the primary antibody or by replacing it with an equivalent concentration of mouse IgG. Nonspecific staining was avoided by careful selection, under the light microscope, of the NGFR-immunostained semithin sections. Controls for the immunocytochemical localization of endogenous NGFR were consistently negative. In control
sections only red blood cells were sometimes stained because of their endogenous peroxidase-like activity.
RESULTS
Light microscopy After immunocytochemistry, N G F R immunoreactivity (-IR) indicating the site of N G F R was detected as a granule-like dark brown reaction product in neurons which had the same morphology and topography as cholinergic basal forebrain magnocellular neurons (Fig. 1A--C). The immunoreaction product was concentrated around the nuclei and also diffusely filled the perikarya and their proximal processes (Fig. IB, inset, C). Patches of immunoreaction product were additionally detected in the outer membrane of the cell body and proximal processes. These patches were particularly evident in osmicated sections (Fig. 1C). Immunoreactive cells were observed in the medial septal nucleus, diagonal band of Broca, substantia innominata and the nucleus basalis magnocellularis (NBM) (Fig. 1A-C). Varicose fibres containing N G F R - I R were occasionally noticed in the NBM. The results of the present study are in good agreement with the observations on the morphology and distribution of NGFR-labelled cells in the rat basal forebrain reported earlier. 3'6'9'12'15-17'24
Electron microscopy Immunoreactive neurons, observed by light microscopy in selected areas of the ventromedial GP, were examined at the electron microscopic level (Figs 1C, D, 3A, B). Electron-dense granular or diffuse immunoreaction product was observed in a number of subcellular organelles which include the Golgi apparatus (GA), rough endoplasmic reticulum ( R E R ) and multivesicular bodies (Figs I D - F , 2 A - D , F , 3A, B). The immunoreactive neurons showed the same cytological features and pattern of synaptic input as those of cholinergic basal forebrain cells. Thus, the cell nucleus showed one or more
Fig. 1. (A, B) Light micrographs illustrating NGFR-positive neurons in the rat basal forebrain immunostained with anti-NGFR (192-IgG) in (A) the nucleus of the diagonal band and (B) area of the NBM. Inset in B is a high power magnification mierograph showing deposits of NGFR-IR within the cytoplasm (denser in the perinuclear area) and along a proximal process of a positive neuron in the NBM. (C, D) Correlated light (C) and electron (D) microscopy of the same NGFR-positive neuron (curved arrow in C) in the ventromedial GP. (C) Light micrograph of a flat embedded, NGFR-immunostained section, postfixed in 1% osmium tetroxide and subsequently processed for electron microscopy. Immunoreaction product had granular appearance (some indicated by arrows) and accumulated mainly in the perinuclear region. The reaction product occupies a proximal process and outlines patches of the plasma membrane of these neurons (crossed arrows). (D) Electron micrograph of the NGFR-positive neuron indicated in C by a curved arrow. Electron-dense reaction product can be observed in multivesicular bodies (arrowheads) and perinucleax Golgi complexes (open arrows). (E) High power magnification electron micrograph of the immunoreactive multivesicular bodies shown in D. (F) High power magnification electron micrograph of an identifed NGFR-positive neuron in the ventromedial GP showing an immunoreactive multivesicular body in the proximity of the cell nucleus (n) and of a Golgi complex exhibiting reaction product in a saccule (arrows). Scale bars for A,B = 100/~m; inset in B and C = 15/zm; D = 0.5/zm; E,F = 0.2/~m.
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Subcellular localization of NGF receptors in the NBM indentations and was surrounded by abundant cytoplasm rich in organelles including lysosomes, mitochondria, RER and GA. Furthermore, the outer membrane of the cell soma and proximal dendrites of NGFR-bearing cells were largely covered by glial processes and also by myelinated and small unmyelinated non-terminal axons (Figs 1D, 3A, E). The glial sheath was penetrated only by a very small number of unlabelled terminal boutons which were presynaptic to NGFR-immunoreactive neurons (Fig. 3E, G). Immunoreaction product was visualized in almost all Golgi configurations (Figs 1D, 3A). Some saecules in the GA were strongly NGFR-positive, whereas others were unreactive (Figs IF, 2A-C, 3B). Deposition of immunoreaction product was seen associated with the internal face of the membrane of the GA saccules. Vesicles still attached to Golgi saccules, or in their proximity, sometimes exhibited N G F R - I R (Fig. 2B). In the cell body, electron-dense immunoreaction was also found associated with membranes of multivesicular bodies (Figs 1D-F, 2C, D). These multivesicular structures contained a number of mostly round vesicles of various sizes and were observed most frequently in the base of neuronal processes (Fig. 1D, E), in the vicinity of the cell nucleus and near the GA (Figs IF, 2C, D). Very rarely, immunoreactivity was observed on small segments of the nuclear envelope (Fig. 2D, E) and in association with the internal face of the membrane of some RER cisternae (Fig. 2F).
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In most magnocellular neurons, N G F R - I R was also associated with the outer surface of the plasma membrane of the soma and proximal dendrites. In the latter, the label was not continuous and diffusely filled the space confined between plasma membranes of immunoreactive neurons and thin glial processes in their vicinity (Fig. 3A-F). These glial processes sometimes exhibited bundles of fibrils, suggesting that they belong to astrocytes (Fig. 3D). Positive immunostaining was also detected associated with the outer surface of the plasma membrane of distal dendrites (Fig. 4A). In the latter, the electron-dense reaction product was localized on structures without visible synaptic specializations diffusely filling the space between apposed plasma membranes of dendrites and glial processes (Fig. 4A). Very occasionally, electrondense immunoreaction product was also detected associated with the internal face of the membrane of a few cisternae of the smooth endoplasmic reticulum of distal dendrites (Fig. 4B). Positive immunostaining was never found associated with synaptic junctions, free in the cytoplasm or within the nucleus, or on the outer surface membrane of subcellular organelles. Distal dendrites that displayed N G F R - I R on the outer surface of the plasma membrane or in the endoplasmic reticulum showed the same synaptic input pattern as cholinergic distal dendrites. They frequently received scarce synaptic input from immunonegative terminals seen in a single plane of a section (Fig. 4) which strongly suggests that they were cholinergic.
Fig. 2. High power magnification electron micrographs showing the localization of NGFR-IR in (A-C) Golgi complexes, (C, D) multivesicular bodies (arrowheads), (D, E) the nuclear envelope (arrows) and (F) associated with the internal surface of membrane of a small segment of a cisternae of the RER (double arrowheads), in identified magnocellular neurons of the ventromedial GP. Note in A-C heavy deposits of reaction product in the lumen of several saccules of the Golgi apparatus (some pointed by open arrows), mostly associated with the internal membrane surface, and in vesiclesstill attached to or in the proximity of the Golgi complex (arrows in B). Also notice in C that the label in the multivesicular body is associated with membranes. (E) Higher magnification of D; the electron-dense reaction product occupies a small segment of the nuclear envelope whereas other cell membranes in the vicinity remain unreactive. Scale bars for A,D=0.2#m; B,C,E,F =0.1 #m. Fig. 3--Overleaf Fig. 3. (A) Electron micrograph of a serial non-adjacent section through the same NGFR-IR neuron shown in Fig. 1D. NGFR-IR can be observed in perinuclear Golgi complexes (open arrows). (B) Magnification of the boxed area in A. Arrows in A (boxed) and B indicate NGFR-IR associated with the outer surface of plasmalemma. Notice in B heavy deposits of electron-dense reaction product in a number of saccules of the Golgi complex (some pointed by open arrows) whereas others stained weakly or remained unreactive (crossed arrows). (C) Higher power magnification electron micrograph of the portion of plasma membrane indicated by arrows in A and B. The electron-dense reaction product (open arrows) fills the space between apposed plasma membranes of the immunoreactive neuron (p) and a glial process (g), reflectingthe extracellular location of the epitope. (D, E) Some examples for the extracellular location of NGFR-IR in identified NGFR-positive magnocellular neurons in the ventromedial GP. Note in D that the reaction product (open arrows) occupies the space between plasma membranes of a neuronal cell body (p) and a thin gliai process (g) exhibiting a bundle of gliofibrils (asterisk). Also notice in E that immunoreactivity (arrows) is located in patches on the neuronal plasma membrane, but is absent from the synaptic cleft of the axosomatic contact (open arrow) established by an immunonegative terminal (t) and the neuron. (F, G) Higher power magnification micrographs showing details of what is shown in E. In F, NGFR-IR (arrowheads) fills the space between plasma membranes of immunoreactive neurons and a thin glial process (open circles). Note that in G, the synaptic cleft of the axosomatic contact (open arrow) is completely free of reaction product. Scale bars for A = I #m; B = 0.2/zm; C = 0.05 #m; D = 0.1 ~um; E = 0.5/zm; F,G =0.1 #m.
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Subcellular localization of NGF receptors in the NBM
Fig. 4. Electron micrographs showing the localization of NGFR-IR (arrows) (A) filling the space between the plasma membranes of a distal dendrite (d) and a glial process (g), probably belonging to an astrocyte, and (B) within a smooth endoplasmic reticulum saccule in a distal dendrite. Note in A and B that the label is not associated with synapses (open arrows). Scale bars for A, B = 0.1 #m.
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The ultrastructure of NGFR-positive neurons of the GP was compared with that of unlabelled cells found in their vicinity. Unlabelled neurons were generally smaller than NGFR-positive cells, had less endoplasmic reticulum, but had many more synaptic contacts on the soma and proximal dendrites. Glial cells within the region of the NBM, in the vicinity of immunoreactive neurons, consistently lacked NGFRIR.
DISCUSSION
The subcellular location of NGFRs in identified cells of the NBM (ventromedial GP) was investigated using 192-IgG. Immunocytochemistry provided: (i) good ultrastructural localization of intracellular NGFR-IR, indicating sites of N G F R synthesis in putative basal forebrain cholinergic neurons; (ii) evidence that NGFRs in these cells never occur in synaptic structures, but rather in non-synaptic neuronal membranes adjacent to astrocytes. Light microscopy The morphology and topography of the NGFRcontaining neurons in the basal forebrain agreed well with previous r e p o r t s . 3'6'9'12,1~17,24 The morphology and distribution of NGFR-immunoreactive neurons resembled that of magnocellular cholinergic neurons of the basal forebrain, supporting the claim that most of NGFR-positive neurons in this region are cholinergic.3,7,13,18 The above, and also the fact that cell bodies and processes of control sections for the immunocytochemical localization of endogenous N G F R were always negative, allowed us the assumption that the immunocytochemical method used in this study produced consistent labelling of the NGFR-bearing neurons in the forebrain of the rat. Electron microscopy At the electron microscopic level, the general appearance of NGFR-positive cells was the same as that of basal forebrain cholinergic neurons, confirming the light microscopic results. The presence of N G F R - I R in the RER, the GA and the external surface of plasmalemma, indicates that NGFRs are synthesized on somatic membrane-bound ribosomes and glycosylated and packed in the GA. The visualization of N G F R IR in the Golgi complex is consistent with the fact that N G F R is a glycoprotein.4'25 From the GA, the receptors are transported to the plasma membrane, where they are inserted to mediate binding of NGF. The detection of N G F R - I R in the endoplasmic reticulum in distal dendrites agrees with a previous study. 16The presence of N G F R - I R in multivesicular bodies (probably lysosomes) may reflect degradation
of N G F - N G F R complexes2~as reported for adrenergic neurons. )4,26 It seems likely that the final processing of endocytic vesicles containing N G F - N G F R complexes includes fusion with multivesicular bodies. Functional considerations Although the biological role and mechanism of action of N G F on cholinergic basal forebrain systems are subjects of great basic and practical interest, they are still poorly understood. N G F specifies differentiation of these neurons and, under certain experimental conditions, plays a neuronotrophic role. These events require receptor-ligand interactions, but the traditional trophic hypothesis requires the neurotrophic ligand to be produced by the neuronal target and taken up at the axonal ending. Therefore, the presence of N G F R in the membranes of soma and dendritic processes of cholinergic neurons remained a puzzle. Here, we have observed that membrane NGFRs are located on neuronal membranes apposed to thin glial processes, but never on synaptic clefts. Based on this observation we proposed that magnocellular basal forebrain neurons may be able to take up N G F via receptors on their cell somata and dendrites. Earlier reports 19'2°'27'33are consistent with a targetderived (hippocampus, neocortex and olfactory bulb) source of the N G F supporting magnocellular cholinergic neurons. It has also been shown 8,1°,23,29 that astrocytes and oligodendrocytes are able to produce N G F in vitro, but the significance of this is unknown. Synthesis of N G F in some areas, like hippocampus, occurs exclusively in neuronsTM whereas in other areas, like the cerebral cortex, both neurons and glial cells appear to be involved in the production of N G F ) ° In the region of the NBM, N G F R - I R selectively deposited in the space between membranes belonging to immunoreactive neurons and thin glial processes, suggests that glia may be a source of NGF. However, there is no evidence that gila make N G F anywhere in the CNS under normal circumstances in vivo.
CONCLUSIONS
The subcellular location of N G F R - I R suggests that: (i) magnocellular basal forebrain cholinergic neurons may be able to take up N G F via receptors on their cell somata and dendrites; (ii) somatic and dendritic NGFRs occur on non-synaptic neuronal membranes, adjacent to astrocytes, and may correspond to low-affinity NGFRs. Acknowledgements--The authors thank Mrs I. Montero, D. Guinea and Mr J. A. Maldonado for excellent technical assistance. This research was supported by DGICYT (grant CSIC 88EA036, SEUI PB0253) and CICYT (grant FAR890683) (Spain).
Subcellular localization of NGF receptors in the NBM
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0306-4522/91 $3.00 + 0.00 Pergamon Press plc © 1991 IBRO
SUBCELLULAR LOCALIZATION OF NERVE GROWTH FACTOR RECEPTORS IN IDENTIFIED CELLS OF THE RAT N U C L E U S BASALIS MAGNOCELLULARIS: A N IMMUNOCYTOCHEMICAL STUDY R. MARTINEz-MuRILLO,*'~ T. FERNANDEZ,I"M. M. ALGUACIL,t F. AGUADO,'~ M. ACHAVAL,~ P. BOVOLENTA,§J. RODRIGO'I"and M. NIETO-SAMPEDRO~ i'Unidad de Neuroanatomia y Unidad de Plasticidad Neuronal, §Instituto Cajal, C.S.I.C., Doctor Arce 37. 28002-Madrid, Spain Abstract--The subcellular location of nerve growth factor receptor in the ventromedial portion of rat globus pallidus was investigated with affinity-purified monoclonal 192-IgG following the unlabelled antibody peroxidase-antiperoxidase immunocytochemical procedure. At the light microscopic level, punctate immunoreaction product was observed in the perinuclear region and in the plasma membrane of large, probably cholinergic neurons. Examination in the electron microscope of these neurons confirmed that nerve growth factor receptor-stained cells were basal forebrain cholinergic neurons. Within these cells, immunostaining occurred in the Golgi apparatus, in multivesieular bodies and, occasionally, in rough endoplasmic reticulum cisternae and the nuclear envelope. Moreover, patches of immunoreactivity were observed associated with the outer surface of the plasma membrane of the soma and their proximal dendrites and also with the plasma membrane of distal dendrites showing scarcity of synaptic input. Positive immunostaining was never observed in synaptic clefts, but filled the space between the plasma membranes of immunoreactive neurons and those of thin glial processes in their vicinity. The location of membrane nerve growth factor receptor in close apposition to membranes of neighbouring astrocytes rather than near synaptic complexes, suggests that glial cells may be a physiological source of nerve growth factor.
s h o w n 3'7'13'18 that a large proportion of magnocellular basal forebrain neurons contains both N G F R and cholinergic enzymes. However, there is little information regarding the subcellular location of the receptor in these cells. 16 Here, we have used an afffinity-purified mouse monoclonal antibody (192IgG) to rat N G F R 5 to elucidate the electron microscopic characteristics of identified N G F R - p o s i t i v e neurons in the ventromedial portion of the globus pallidus (GP), and to visualize the subcellular location of N G F R in these cells.
Nerve growth factor ( N G F ) is a well-characterized neuronotrophic protein essential for the development, survival and maintenance of function of sympathetic- and neural crest-derived sensory neurons in the peripheral nervous system (PNS) (for reviews see Refs 11,22, 31) and of cholinergic neurons in the C N S (for review see Refs 30, 35). It also plays a critical role regulating the size of cell body, process extension and content of selected proteins, including choline acetyltransferase (CHAT), acetylcholinesterase (ACHE) and nerve growth factor receptor ( N G F R ) , of adult septal cholinergic neurons after axotomy. ~2 The presence of N G F R in basal forebrain magnocellular neurons of both rat and human has been amply documented at the light microscopic level. 1'3'6'9'13'15"17'24'28'36 Moreover, it has also been
EXPERIMENTAL PROCEDURES
Animals and tissue preparation
*To whom correspondence should be addressed. :~Present address: Departamento de Cirncias Morfol6gicas, Instituto de Biocirncias, Universidade Federal do Rio Grande do Sul. Porto Alegre, Brasil. Abbreviations: ACHE, acetylcholinesterase; CHAT, choline acetyltransferase; DAB, 3,3'-diaminobenzidine; GA, Golgi apparatus; GP, globus pallidus; NBM, nucleus basalis magnocellularis; NGF, nerve growth factor; NGFR(s), nerve growth factor receptors; NGFR-IR, nerve growth factor receptor immunoreactivity; PAP, peroxidase-antiperoxidase; PB, phosphate buffer; PBS, phosphate-buffered saline; PNS, peripheral nervous system; RER, rough endoplasmic reticulum. 463
Experiments were performed on six adult, male, albino Wistar rats (Charles River) of approximately 250 g. Under deep anaesthesia (Equithesin, Jansen Lab., 2.5 ml/kg intraperitoneally), animals were ventilated and perfused through the aorta with 0.9% saline followed by 400 ml of a fixative solution containing 4% paraformaldehyde, 0.1% glutaraldehyde and 0.03% CaC12 in 0.1 M phosphate buffer (PB) pH 7.4. Perfused brains were removed and cut into small blocks. Blocks with the selected areas were postfixed for 4h (4% paraformaldehyde in 0.1 M PB) at room temperature and cryoprotected by immersion in 30% sucrose solution in 0.1 M PB at 4°C. Nerve growth factor receptor immunocytochemistry Light microscopy. Studies were performed on two animals. After fixation and cryoprotection, serial 40-/~m-thick
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frontal frozen sections were prepared with a Leitz sledge microtome. Sections were rinsed in phosphate-buffered saline (PBS) and processed for NGFR immunocytochemistry using affinity-purified mouse monoclonal antibody (192-IgG) to rat NGFR, 5 following the peroxidaseantiperoxidase (PAP) procedure. Free-floating sections were incubated in the primary antibody (final concentration 5 #g/ml, in PBS containing 0.01% sodium azide and 0.2% Triton X-100), overnight with continuous agitation at 4°C. The remaining operations were performed at room temperature. Tissue sections were washed several times in PBS, incubated with rabbit anti-mouse IgG (Biomakor, bm; 1: 10 in PBS) for 1 h, washed again in PBS and incubated in monoclonal mouse PAP antibody (Biomakor, bm; 1: 1000 in PBS) for 90 min. The immunocytochemical reaction was revealed by incubating the tissue sections in a histochemical medium that contained 0.06% 3,Y-diaminobenzidine (DAB) tetra HC1 (Sigma, St Louis, MO) in PBS, for I0 min, and then in the same solution containing 0.003% H202. The DAB reaction was interrupted at times chosen by inspection of trial sections (approximately 8 min). Electron microscopy. Electron microscopic studies were performed on four animals. Cryoprotected blocks were rapidly frozen with liquid nitrogen and thawed in cold 0.1 M PB to improve the antibody penetration. Sections (40/~m thick) were cut with a Vibratome (Lancer) and immunostained as for light microscopy except that Triton X-100 was not included in the incubation solutions. The double bridge procedure 32 was used and incubations with rabbit antimouse IgG and mouse PAP were repeated, after washing, using the same dilutions of reagents as were used in the first bridging step (30 min each). After the DAB reaction, sections were washed in PBS (I h), postfixed in 1% osmium tetroxide in 0.1 M PB, pH 7.4 (1 h), dehydrated in ethanols and block-stained in uranyl acetate (1% in 70% ethanol) in the dark for 40 min at room temperature. The sections were mounted on Durcupan ACM resin (Fluka) slides under a plastic coverslip and cured for three days at 56°C. Selected areas of the ventromedial part of the GP, containing NGFR-positive neurons, were dissected out, re-embedded in Durcupan in plastic capsules and semithin sections (2-2.5/~m thick) were then prepared. Selected semithin sections were re-embedded in Durcupan in plastic capsules. Ultrathin serial sections were then obtained, mounted on Formvar-coated grids, stained with lead citrate and examined in a Jeol 1200 EX electron microscope. Control sections were prepared either by omitting the primary antibody or by replacing it with an equivalent concentration of mouse IgG. Nonspecific staining was avoided by careful selection, under the light microscope, of the NGFR-immunostained semithin sections. Controls for the immunocytochemical localization of endogenous NGFR were consistently negative. In control
sections only red blood cells were sometimes stained because of their endogenous peroxidase-like activity.
RESULTS
Light microscopy After immunocytochemistry, N G F R immunoreactivity (-IR) indicating the site of N G F R was detected as a granule-like dark brown reaction product in neurons which had the same morphology and topography as cholinergic basal forebrain magnocellular neurons (Fig. 1A--C). The immunoreaction product was concentrated around the nuclei and also diffusely filled the perikarya and their proximal processes (Fig. IB, inset, C). Patches of immunoreaction product were additionally detected in the outer membrane of the cell body and proximal processes. These patches were particularly evident in osmicated sections (Fig. 1C). Immunoreactive cells were observed in the medial septal nucleus, diagonal band of Broca, substantia innominata and the nucleus basalis magnocellularis (NBM) (Fig. 1A-C). Varicose fibres containing N G F R - I R were occasionally noticed in the NBM. The results of the present study are in good agreement with the observations on the morphology and distribution of NGFR-labelled cells in the rat basal forebrain reported earlier. 3'6'9'12'15-17'24
Electron microscopy Immunoreactive neurons, observed by light microscopy in selected areas of the ventromedial GP, were examined at the electron microscopic level (Figs 1C, D, 3A, B). Electron-dense granular or diffuse immunoreaction product was observed in a number of subcellular organelles which include the Golgi apparatus (GA), rough endoplasmic reticulum ( R E R ) and multivesicular bodies (Figs I D - F , 2 A - D , F , 3A, B). The immunoreactive neurons showed the same cytological features and pattern of synaptic input as those of cholinergic basal forebrain cells. Thus, the cell nucleus showed one or more
Fig. 1. (A, B) Light micrographs illustrating NGFR-positive neurons in the rat basal forebrain immunostained with anti-NGFR (192-IgG) in (A) the nucleus of the diagonal band and (B) area of the NBM. Inset in B is a high power magnification mierograph showing deposits of NGFR-IR within the cytoplasm (denser in the perinuclear area) and along a proximal process of a positive neuron in the NBM. (C, D) Correlated light (C) and electron (D) microscopy of the same NGFR-positive neuron (curved arrow in C) in the ventromedial GP. (C) Light micrograph of a flat embedded, NGFR-immunostained section, postfixed in 1% osmium tetroxide and subsequently processed for electron microscopy. Immunoreaction product had granular appearance (some indicated by arrows) and accumulated mainly in the perinuclear region. The reaction product occupies a proximal process and outlines patches of the plasma membrane of these neurons (crossed arrows). (D) Electron micrograph of the NGFR-positive neuron indicated in C by a curved arrow. Electron-dense reaction product can be observed in multivesicular bodies (arrowheads) and perinucleax Golgi complexes (open arrows). (E) High power magnification electron micrograph of the immunoreactive multivesicular bodies shown in D. (F) High power magnification electron micrograph of an identifed NGFR-positive neuron in the ventromedial GP showing an immunoreactive multivesicular body in the proximity of the cell nucleus (n) and of a Golgi complex exhibiting reaction product in a saccule (arrows). Scale bars for A,B = 100/~m; inset in B and C = 15/zm; D = 0.5/zm; E,F = 0.2/~m.
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Subcellular localization of NGF receptors in the NBM indentations and was surrounded by abundant cytoplasm rich in organelles including lysosomes, mitochondria, RER and GA. Furthermore, the outer membrane of the cell soma and proximal dendrites of NGFR-bearing cells were largely covered by glial processes and also by myelinated and small unmyelinated non-terminal axons (Figs 1D, 3A, E). The glial sheath was penetrated only by a very small number of unlabelled terminal boutons which were presynaptic to NGFR-immunoreactive neurons (Fig. 3E, G). Immunoreaction product was visualized in almost all Golgi configurations (Figs 1D, 3A). Some saecules in the GA were strongly NGFR-positive, whereas others were unreactive (Figs IF, 2A-C, 3B). Deposition of immunoreaction product was seen associated with the internal face of the membrane of the GA saccules. Vesicles still attached to Golgi saccules, or in their proximity, sometimes exhibited N G F R - I R (Fig. 2B). In the cell body, electron-dense immunoreaction was also found associated with membranes of multivesicular bodies (Figs 1D-F, 2C, D). These multivesicular structures contained a number of mostly round vesicles of various sizes and were observed most frequently in the base of neuronal processes (Fig. 1D, E), in the vicinity of the cell nucleus and near the GA (Figs IF, 2C, D). Very rarely, immunoreactivity was observed on small segments of the nuclear envelope (Fig. 2D, E) and in association with the internal face of the membrane of some RER cisternae (Fig. 2F).
467
In most magnocellular neurons, N G F R - I R was also associated with the outer surface of the plasma membrane of the soma and proximal dendrites. In the latter, the label was not continuous and diffusely filled the space confined between plasma membranes of immunoreactive neurons and thin glial processes in their vicinity (Fig. 3A-F). These glial processes sometimes exhibited bundles of fibrils, suggesting that they belong to astrocytes (Fig. 3D). Positive immunostaining was also detected associated with the outer surface of the plasma membrane of distal dendrites (Fig. 4A). In the latter, the electron-dense reaction product was localized on structures without visible synaptic specializations diffusely filling the space between apposed plasma membranes of dendrites and glial processes (Fig. 4A). Very occasionally, electrondense immunoreaction product was also detected associated with the internal face of the membrane of a few cisternae of the smooth endoplasmic reticulum of distal dendrites (Fig. 4B). Positive immunostaining was never found associated with synaptic junctions, free in the cytoplasm or within the nucleus, or on the outer surface membrane of subcellular organelles. Distal dendrites that displayed N G F R - I R on the outer surface of the plasma membrane or in the endoplasmic reticulum showed the same synaptic input pattern as cholinergic distal dendrites. They frequently received scarce synaptic input from immunonegative terminals seen in a single plane of a section (Fig. 4) which strongly suggests that they were cholinergic.
Fig. 2. High power magnification electron micrographs showing the localization of NGFR-IR in (A-C) Golgi complexes, (C, D) multivesicular bodies (arrowheads), (D, E) the nuclear envelope (arrows) and (F) associated with the internal surface of membrane of a small segment of a cisternae of the RER (double arrowheads), in identified magnocellular neurons of the ventromedial GP. Note in A-C heavy deposits of reaction product in the lumen of several saccules of the Golgi apparatus (some pointed by open arrows), mostly associated with the internal membrane surface, and in vesiclesstill attached to or in the proximity of the Golgi complex (arrows in B). Also notice in C that the label in the multivesicular body is associated with membranes. (E) Higher magnification of D; the electron-dense reaction product occupies a small segment of the nuclear envelope whereas other cell membranes in the vicinity remain unreactive. Scale bars for A,D=0.2#m; B,C,E,F =0.1 #m. Fig. 3--Overleaf Fig. 3. (A) Electron micrograph of a serial non-adjacent section through the same NGFR-IR neuron shown in Fig. 1D. NGFR-IR can be observed in perinuclear Golgi complexes (open arrows). (B) Magnification of the boxed area in A. Arrows in A (boxed) and B indicate NGFR-IR associated with the outer surface of plasmalemma. Notice in B heavy deposits of electron-dense reaction product in a number of saccules of the Golgi complex (some pointed by open arrows) whereas others stained weakly or remained unreactive (crossed arrows). (C) Higher power magnification electron micrograph of the portion of plasma membrane indicated by arrows in A and B. The electron-dense reaction product (open arrows) fills the space between apposed plasma membranes of the immunoreactive neuron (p) and a glial process (g), reflectingthe extracellular location of the epitope. (D, E) Some examples for the extracellular location of NGFR-IR in identified NGFR-positive magnocellular neurons in the ventromedial GP. Note in D that the reaction product (open arrows) occupies the space between plasma membranes of a neuronal cell body (p) and a thin gliai process (g) exhibiting a bundle of gliofibrils (asterisk). Also notice in E that immunoreactivity (arrows) is located in patches on the neuronal plasma membrane, but is absent from the synaptic cleft of the axosomatic contact (open arrow) established by an immunonegative terminal (t) and the neuron. (F, G) Higher power magnification micrographs showing details of what is shown in E. In F, NGFR-IR (arrowheads) fills the space between plasma membranes of immunoreactive neurons and a thin glial process (open circles). Note that in G, the synaptic cleft of the axosomatic contact (open arrow) is completely free of reaction product. Scale bars for A = I #m; B = 0.2/zm; C = 0.05 #m; D = 0.1 ~um; E = 0.5/zm; F,G =0.1 #m.
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Subcellular localization of NGF receptors in the NBM
Fig. 4. Electron micrographs showing the localization of NGFR-IR (arrows) (A) filling the space between the plasma membranes of a distal dendrite (d) and a glial process (g), probably belonging to an astrocyte, and (B) within a smooth endoplasmic reticulum saccule in a distal dendrite. Note in A and B that the label is not associated with synapses (open arrows). Scale bars for A, B = 0.1 #m.
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The ultrastructure of NGFR-positive neurons of the GP was compared with that of unlabelled cells found in their vicinity. Unlabelled neurons were generally smaller than NGFR-positive cells, had less endoplasmic reticulum, but had many more synaptic contacts on the soma and proximal dendrites. Glial cells within the region of the NBM, in the vicinity of immunoreactive neurons, consistently lacked NGFRIR.
DISCUSSION
The subcellular location of NGFRs in identified cells of the NBM (ventromedial GP) was investigated using 192-IgG. Immunocytochemistry provided: (i) good ultrastructural localization of intracellular NGFR-IR, indicating sites of N G F R synthesis in putative basal forebrain cholinergic neurons; (ii) evidence that NGFRs in these cells never occur in synaptic structures, but rather in non-synaptic neuronal membranes adjacent to astrocytes. Light microscopy The morphology and topography of the NGFRcontaining neurons in the basal forebrain agreed well with previous r e p o r t s . 3'6'9'12,1~17,24 The morphology and distribution of NGFR-immunoreactive neurons resembled that of magnocellular cholinergic neurons of the basal forebrain, supporting the claim that most of NGFR-positive neurons in this region are cholinergic.3,7,13,18 The above, and also the fact that cell bodies and processes of control sections for the immunocytochemical localization of endogenous N G F R were always negative, allowed us the assumption that the immunocytochemical method used in this study produced consistent labelling of the NGFR-bearing neurons in the forebrain of the rat. Electron microscopy At the electron microscopic level, the general appearance of NGFR-positive cells was the same as that of basal forebrain cholinergic neurons, confirming the light microscopic results. The presence of N G F R - I R in the RER, the GA and the external surface of plasmalemma, indicates that NGFRs are synthesized on somatic membrane-bound ribosomes and glycosylated and packed in the GA. The visualization of N G F R IR in the Golgi complex is consistent with the fact that N G F R is a glycoprotein.4'25 From the GA, the receptors are transported to the plasma membrane, where they are inserted to mediate binding of NGF. The detection of N G F R - I R in the endoplasmic reticulum in distal dendrites agrees with a previous study. 16The presence of N G F R - I R in multivesicular bodies (probably lysosomes) may reflect degradation
of N G F - N G F R complexes2~as reported for adrenergic neurons. )4,26 It seems likely that the final processing of endocytic vesicles containing N G F - N G F R complexes includes fusion with multivesicular bodies. Functional considerations Although the biological role and mechanism of action of N G F on cholinergic basal forebrain systems are subjects of great basic and practical interest, they are still poorly understood. N G F specifies differentiation of these neurons and, under certain experimental conditions, plays a neuronotrophic role. These events require receptor-ligand interactions, but the traditional trophic hypothesis requires the neurotrophic ligand to be produced by the neuronal target and taken up at the axonal ending. Therefore, the presence of N G F R in the membranes of soma and dendritic processes of cholinergic neurons remained a puzzle. Here, we have observed that membrane NGFRs are located on neuronal membranes apposed to thin glial processes, but never on synaptic clefts. Based on this observation we proposed that magnocellular basal forebrain neurons may be able to take up N G F via receptors on their cell somata and dendrites. Earlier reports 19'2°'27'33are consistent with a targetderived (hippocampus, neocortex and olfactory bulb) source of the N G F supporting magnocellular cholinergic neurons. It has also been shown 8,1°,23,29 that astrocytes and oligodendrocytes are able to produce N G F in vitro, but the significance of this is unknown. Synthesis of N G F in some areas, like hippocampus, occurs exclusively in neuronsTM whereas in other areas, like the cerebral cortex, both neurons and glial cells appear to be involved in the production of N G F ) ° In the region of the NBM, N G F R - I R selectively deposited in the space between membranes belonging to immunoreactive neurons and thin glial processes, suggests that glia may be a source of NGF. However, there is no evidence that gila make N G F anywhere in the CNS under normal circumstances in vivo.
CONCLUSIONS
The subcellular location of N G F R - I R suggests that: (i) magnocellular basal forebrain cholinergic neurons may be able to take up N G F via receptors on their cell somata and dendrites; (ii) somatic and dendritic NGFRs occur on non-synaptic neuronal membranes, adjacent to astrocytes, and may correspond to low-affinity NGFRs. Acknowledgements--The authors thank Mrs I. Montero, D. Guinea and Mr J. A. Maldonado for excellent technical assistance. This research was supported by DGICYT (grant CSIC 88EA036, SEUI PB0253) and CICYT (grant FAR890683) (Spain).
Subcellular localization of NGF receptors in the NBM
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