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.
The cellular distribution and functional aspects of neurotensin (NT) binding sites in rat mesencephalic cells in primary culture were investigated by an original approach combining anatomical and biochemical studies. Using a double-labeling protocol combining 125I-NT receptor radioautography and tyrosine hydroxylase (TH) immunocytochemistry, we obtained the first direct visualization of NT binding sites on TH-immunoreactive neurons. Eighty percent of the TH neurons were endowed with NT binding sites, which can be observed on both cell bodies and processes. TH-immunoreactive neurons were characterized as dopaminergic neurons by their ability to take up dopamine in a benztropine- and nomifensine-sensitive manner. In the mesencephalic cultures, NT increased potassium-evoked release of tritiated dopamine, and the relative potencies of various NT-related peptides to increase dopamine release were in good agreement with their abilities to bind to NT sites. These results show for the first time that cultured rat mesencephalic dopaminergic cells express functional NT receptors. Finally, the specificity and distribution of NT receptors on dopaminergic neurons in primary culture are quite similar to what was observed in the adult rat brain using pharmacological and radioautographic approaches. These data indicate that NT can influence the activity of dopaminergic neurons at very early stages of the rat brain development.
In the rat brain, destruction of dopaminergic cell groups by injections of 6-hydroxydopamine into the ventral mesencephalic tegmentum results in large decreases in the number of neurotensin binding sites in the mesencephalon and the striatum. In contrast, these lesions produce an increase in the number of 1251-labeled neurotensin binding sites in the lateral part of the prefrontal cortex despite a large decrease in cortical dopamine levels. Increases; in the number of 125I-labeled neurotensin binding sites in this cortical area as well as in the entorhinal cortex, the nucleus accumbens, and the central part of the striatum were also obtained after chronic blockade of dopamine neurotransmission by a long-acting neuroleptic pipotiazine palmitic ester. We propose that dopamine inputs regulate the density of postsynaptic neurotensin binding sites through cortical and subcortical dopamine receptors. Therefore, some of the clinical effects of neuroleptics in schizophrenic patients could be partly related to changes in neurotensin neurotransmission.Neurotensin (NT) is a tridecapeptide that is unevenly distributed in mammalian brain (1-4). High levels of NT immunoreactivity have been found in some structures innervated by ascending dopaminergic neurons, such as the nucleus accumbens or the striatum, while relatively little NT immunoreactivity was detected in others, such as the prefrontal cortex (5, 6). Histochemical studies have also revealed the occurrence of NT immunoreactive fibers in the ventral mesencephalic tegmentum (VMT) and in the zona compacta and pars lateralis of the substantia nigra (SN) surrounding dopamine-containing cell bodies and their dendrites (7). In addition, numerous neuronal perikarya of the VMT contain NT immunoreactivity: NT and dopamine coexist in some VMT neurons (7) and the existence of a VMT NT projection to the median nucleus accumbens has been also described (8).There is some indication that NT stimulates the firing rate of nigral dopaminergic cells (9), and several biochemical and behavioral studies have suggested that intraventricularly or locally injected NT can activate dopaminergic cells located either in the VMT or in the SN (10-14). For example, in the rat, the injection of NT into the VMT produced concomitant increases in the levels of dihydroxyphenylacetic acid in the nucleus accumbens and in locomotor activity (13 Although interactions between NT and dopaminergic neurons are well-documented, there is no report about the influence of dopamine neurotransmission on the NT binding sites located post-synaptically to the dopaminergic neurons. In the present study, two approaches were used to chronically interrupt dopamine transmission: the first consisted of the destruction of ascending dopaminergic neurons by bilateral injections of 6-hydroxydopamine into the VMT, and the second took advantage of the property of a long-acting neuroleptic, the pipotiazine palmitic ester, to chronically block dopamine receptors. Changes in NT binding sites were examined by using in vitro qua...
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