Electron microscopic localization of neurotensin binding sites in the midbrain tegmentum of the rat. I. Ventral tegmental area and the interfascicular nucleus

Abstract

The distribution of specifically labeled neurotensin (NT) binding sites was examined by light and electron microscopic radioautography in the ventral tegmental area (VTA) and nucleus interfascicularis of the rat following incubation of lightly prefixed midbrain slices with the monoiodinated ligand, 125I-(Tyr3)-NT. Film radioautograms of whole 125I-NT-incubated slices exhibited intense NT displaceable binding throughout the VTA and interfascicular nucleus. In light microscopic radioautographs from 1-microns-thick sections taken from the surface of the slices, the label was found to be present both inside and outside neuronal perikarya. Probability circle analysis of silver grain distribution in electron microscopic radioautographs confirmed that a significant proportion (greater than 20%) of the specifically labeled binding sites was intraneuronal. The frequent association of these sites with profiles of rough endoplasmic reticulum or Golgi apparatus suggested that they corresponded in part to receptors undergoing synthesis and/or glycosylation. The remainder was associated with neuronal and/or glial plasma membranes, as attested by comparing the distribution of grains overlying apposed cellular elements with the distribution of hypothetical grains originating from randomly distributed membrane bound radioactive sources. Although the resolution of the technique did not make it possible to ascribe labeled membrane-bound receptors to either one of the apposed plasma membranes, their frequent association with interfaces involving the plasmalemma of perikarya and dendrites, together with the occurrence of silver grain alignments along the membrane of certain somata and dendrites suggested that a proportion of them was associated with the perikarya and dendrites of a subpopulation of ventral tegmental neurons. Interestingly, these perikaryal and dendritic receptors were not exclusively present on, or even concentrated opposite, abutting axon terminals but instead were more or less evenly distributed along the plasma membrane. Only an exceedingly small proportion was found to be associated with synaptic junctions. Such a low incidence makes it unlikely that only the synapse-linked binding sites correspond to functional receptors. On the contrary, the dispersion of labeled receptors seen here along the plasma membrane of presumptive dopamine neurons suggests that NT acts mainly in a paracrine or parasynaptic fashion in the ventral midbrain tegmentum.

Complementarity of radioautographic and immunohistochemical techniques for localizing neuroreceptors at the light and electron microscopy level

“…Such a homogeneous distribution is consistent with the earlier demonstration that, within the substantia nigra, neurotensin-containing axon terminals only rarely contact neurotensin receptor-bearing (i.e., dopaminergic) cells (34). It is also congruent with the previously proposed hypothesis that throughout several regions of the brain, including the substantia nigra (34), ventral tegmental area (14) and basal forebrain (35), neurotensin acts in a parasynaptic manner by diffusion for short distances into the extracellular space.…”

“…The introduction of photoaffinity probes and of radioligands that lent themselves to crosslinking by divalent agents made it possible to visualize covalently labeled receptors by means of standard wet autoradiographic techniques (6,7). This technical improvement significantly increased the resolution of receptor detection allowing their visualization at both the cellular (e.g., [8][9][10][11] and subcellular levels (7,(12)(13)(14)(15).…”

From the Golgi–Cajal mapping to the transmitter-based characterization of the neuronal networks leading to two modes of brain communication: Wiring and volume transmission

“…The ultrastructural analysis based on transmitter and receptor immunocytochemistry has repeatedly demonstrated short distance transmitter/receptor mismatches for the peptide and classical transmitters with the receptor labeling outside synapses (Dana et al, 1989;Pasquini et al, 1992;Levey et al, 1993;Liu et al, 1994;Sesack et al, 1994;Aoki, 1992;Aoki and Pickel, 1992;Yung et al, 1995;Caillé et al, 1996;Aoki et al, 1998;Azmitia and Whitaker-Azmitia, 1991;Azmitia et al, 1996;Boudin et al, 1998;Baude and Shigemoto, 1998;Dournaud et al, 1998). Extrasynaptic location of receptors has been observed mainly for G-protein-coupled receptors, but also for ion channel linked receptors .…”

Neurotensin excitation of rat ventral tegmental neurones.

“…Thus, neurotensin is colocalized with dopamine in cells of the arcuate nucleus, ventral tegmental area and substantia nigra (H6kfelt, Everitt, Theodorsson-Norhedin & Goldstein, 1984;Bayer, Towle & Pickel, 1991), and neurotensin and dopamine are released together from terminals in the prefrontal cortex (Bean, During & Roth, 1989). Neurotensin binding sites are to be found in the highest density either in the prefrontal cortex or in these dopamine-containing cell body regions (Young & Kuhar, 1981;Kanba, Kanba, Okazaki & Richelson, 1986); in the latter case, the receptor is on the dopamine-containing neurones (Szigethy & Beaudet, 1989;Dana et al 1989). Neurotensin increases firing of these cells when recorded in vivo or in vitro (Pinnock, 1985;Seutin, Massotte & Dresse, 1989).…”