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. 1998 Oct 15;18(20):8473-84.
doi: 10.1523/JNEUROSCI.18-20-08473.1998.

Correlative ultrastructural distribution of neurotensin receptor proteins and binding sites in the rat substantia nigra

H Boudin  1 , D Pélaprat, W Rostène, V M Pickel, A Beaudet
Affiliations

Correlative ultrastructural distribution of neurotensin receptor proteins and binding sites in the rat substantia nigra

H Boudin et al. J Neurosci. .

Abstract

Neurotensin (NT) produces various stimulatory effects on dopaminergic neurons of the rat substantia nigra. To gain insight into the subcellular substrate for these effects, we compared by electron microscopy the distribution of immunoreactive high-affinity NT receptor proteins (NTRH) with that of high-affinity 125I-NT binding sites in this region of rat brain. Quantitative analysis showed a predominant association of immunogold and radioautographic labels with somata and dendrites of presumptive dopaminergic neurons, and a more modest localization in myelinated and unmyelinated axons and astrocytic leaflets. The distributions of immunoreactive NTRH and 125I-NT binding sites along somatodendritic plasma membranes were highly correlated and homogeneous, suggesting that membrane-_targeted NTRH proteins were functional and predominantly extrasynaptic. Abundant immunocytochemically and radioautographically labeled receptors were also detected inside perikarya and dendrites. Within perikarya, these were found in comparable proportions over membranes of smooth endoplasmic reticulum and Golgi apparatus, suggesting that newly synthesized receptor proteins already possess the molecular and conformational properties required for effective ligand binding. By contrast, dendrites showed a proportionally higher concentration of immunolabeled than radiolabeled intracellular receptors. A fraction of these immunoreactive receptors were found in endosomes, suggesting that they had undergone ligand-induced internalization and were under a molecular conformation and/or in a physical location that precluded their recognition by and/or access to exogenous ligand. Our results provide the first evidence that electron microscopic immunocytochemistry of the NT receptor identifies sites for both the binding and trafficking of NT in the substantia nigra.

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Figures

Fig. 1.
Fig. 1.
Light microscopic distribution of NTRH immunoreactivity (a, a′) and radioautographic distribution of bound 125I-NT (b, b′) in the rat ventral mesencephalon. Sections 40 and 75 μm thick, respectively, were processed using immunoperoxidase (a, a′) and “dry” radioautography (b, b′). For both markers, specific signal is prominent in the substantia nigra, pars compacta (SNC), and ventral tegmental area (VTA), with dendrites extending in the pars reticulata (SNR;arrows). Note the lower sensitivity but higher resolution of the histochemical, as compared with autoradiographic, signal. Control sections immunoreacted with Abi3 antiserum preadsorbed with 0.1 μg/ml of antigenic peptide (a′) or incubated with 125I-NT in the presence of 500 nmnonradioactive NT (b′) are totally devoid of labeling. Scale bars, 500 μm.
Fig. 2.
Fig. 2.
Quantitative analysis of the ultrastructural distribution of immunoreactive NTRH receptors detected inside or on the plasma membrane of dendrites, somas, axon terminals, unmyelinated axons (Axon), myelinated axons, and glial cells in the substantia nigra, pars compacta. Percentages (mean ± SEM) are based on the number of gold particles observed in ultrathin sections from three animals (out of a total of 650 particles/animal).
Fig. 3.
Fig. 3.
Electron micrographs showing immunoreactivity of immunogold-labeled NTRH receptors in neuronal cell bodies (a, b), myelinated axons, and dendrite (c).a, b, Within neuronal cell bodies, gold particles are seen in association with endoplasmic reticulum (ER), Golgi apparatus (G), and tubulovesicles (TV) located in the vicinity of the Golgi apparatus. c, Although immunogold particles associated with myelinated axons are predominantly intracytoplasmic, those associated with the dendritic shaft are all on the plasma membrane. Scale bars, 0.6 μm.
Fig. 4.
Fig. 4.
Dendritic shafts immunolabeled for NTRH (a) and radioautographically labeled for125I-NT binding sites (b, c). Ina, one of the dendrites (D1) shows only intracellular labeling; the other (D2) is labeled predominantly on its plasma membrane. Three gold particles are found opposite unmyelinated and one opposite myelinated axons (ma). In b, resolutioncircles of the size used for statistical distribution analysis help to differentiate exclusive (intracellular;arrows) from shared (membrane-associated) labeled sites. This labeled profile exhibits a single shared grain at the level of an abutting axon terminal (*). In c, all grains decorate the plasma membrane of the labeled dendrite. One of these encroaches on the plasma membrane of an adjacent axon terminal (arrow). Scale bars: a, 0.6 μm;b, c, 1 μm.
Fig. 5.
Fig. 5.
NTRH proteins (a, c) and125I-NT binding sites (b, d) associated with dendritic plasma membranes in rat substantia nigra. In the top panels, gold particles (a) as well as radioautographic grains (b) are detected over axodendritic contacts. In a, an asymmetric synaptic specialization is clearly visible at the site of contact (arrow). In b, there is no obvious synaptic specialization in the plane of section. Both abutting terminals contain densely packed clear synaptic vesicles. Ina, note the high density of gold particles associated with intradendritic vesicles (arrowhead). In the bottom panels, gold particles (c) and radioautographic grains (d) associated with dendritic plasma membranes are detected opposite thin astroglial sheaths. Scale bars, 0.5 μm.
Fig. 6.
Fig. 6.
Distribution of NTR immunoreactivity (a) and of specific 125I-NT binding (b) sites along dendritic plasma membranes.a, The proportion of dendritic membrane-associated gold particles facing each type of abutting element is expressed as a percentage of the total number of gold particles associated with dendritic membranes. The proportion of membrane length occupied by these different elements is expressed as a percentage of total dendritic perimeter. Values are the mean ± SEM from three animals. b, The distribution of specific125I-NT binding sites along dendritic plasma membrane is derived from the labeling frequency of membrane interfaces involving dendrites and expressed as a percentage of shared grains associated with dendrites. The frequency with which dendritic membranes were contacted by its different abutting elements is similarly derived from the occurrence frequency of hypothetical shared grains overlying membrane interfaces involving dendrites. Values are the mean ± SEM of six sections from four animals.
Fig. 7.
Fig. 7.
Intracellular distribution of NTRH proteins (gold particles) and 125I-NT binding sites (exclusive silver grains) in rat substantia nigra. Data are expressed as percentage of the total number of intracellular gold particles for NTRH and of the total number of exclusive grains for 125I-NT binding sites. Values are the mean ± SEM from three (immunocytochemistry; total particles counted: 1950) and four (radioautography; total grains counted: 1632) animals.
Fig. 8.
Fig. 8.
Intrasomatic distribution of NTRH receptor proteins (gold particles) and 125I-NT binding sites (silver grains) in rat substantia nigra. The number of gold particles and of silver grains associated with endoplasmic reticulum (ER), Golgi apparatus (Golgi), mitochondria, vesicles, and nucleus was recorded and expressed as a percentage of total intracellular somatic labeling. Values are the mean ± SEM from three (immunocytochemistry) and four (radioautography) animals.
Fig. 9.
Fig. 9.
NTRH-immunoreactive dendrites in rat substantia nigra. Intracellular receptors. a, Two NTRH-immunoreactive dendritic shafts (D1, D2) in which immunolabeled receptors are exclusively intracellular. In D1, gold particles are associated with the outer limiting membrane of large endocytic vesicles (arrows). In D2, immunogold particles are associated with a mitochondrion (arrowhead) or with small, secretory-type vesicles (arrows).b, Two NTRH-immunoreactive shafts (D3, D4), one of which (D4) receives a symmetrical synaptic contact from an unlabeled terminal (arrowhead). In both dendrites, immunogold particles are seen in association with the outer membrane of endosomal vesicles (arrows). Note that the vesicle facing the axon terminal is in the process of endocytosis (large arrow).c, A single NTRH-labeled dendrite showing intracellular receptors associated either with a large, endocytotic-type vesicle (arrow) or with a small, secretory-type vesicle (arrowhead). Scale bars: a, b, 0.5 μm;c, 0.2 μm.
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